Elisa Delia De Vivo

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.

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.

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.

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.

Ritonavir-boosted darunavir (DRV/r) and dolutegravir (DTG) are affected by induction of metabolizing enzymes and efflux transporters caused by rifampicin (RIF). This complicates the treatment of people living with HIV (PLWH) diagnosed with tuberculosis. Recent data showed that doubling DRV/r dose did not compensate for this effect, and hepatic safety was unsatisfactory.

Background: Antiretroviral therapy reduces systemic inflammation and immune activation, but not to levels like HIV-negative. Limited drug penetration within tissues has been argued as potential mechanism of persistent inflammation. Data on the role of inflammation on plasma/intracellular (IC) pharmacokinetics (PK) of ARV drugs through to downregulation/expression of cytochrome P450 3A/membrane transport proteins are limited. Aim of this study was to investigate the correlation between inflammation markers and plasma/IC PK of different ARVs regimen in HIV-positive patients. Methods: We included in the study ART-treated HIV+ pts switching to 3 different ARV regimens: 1) DTG-based dual-therapy plus boosted-PIs, 2) DTG-based triple-therapy without PIs, 3) DRV/c-based triple-therapy. Plasma and IC ARV drugs concentration means at the end of dosing interval (T0), IM on samples concomitantly with ARV PK determination: sCD14, CRP, IL-6 and LPS were analysed. Results: 60 samples from pts included in the switching study were used for measuring plasma and IC concentrations of HIV drugs. No significative differences between CRP, sCD14, IL-6 and LPS values in 3 arms of therapy were observed. Significant correlation was observed between tenofovir plasma concentrations and sCD14 (p<0.001), DRV plasma concentration and sCD14 (p=0,07) and DRV IC/plasma ratio and Log10 IL-6 concentrations (p=0.04). Furthermore, in 24 pts on DTG-TT, we observed a negative trend between DTG IC concentrations and sCD14 (p=0.09). Conclusions: Our preliminary data support the hypothesis of lower IC concentrations of DRV and DTG in pts with higher plasma IM, suggesting an interplay between HIV drug penetration and persistent inflammation in cART-treated HIV-positive patients.

Dalbavancin (DBV) is an intravenous long-acting second-generation glycolipopeptide antibiotic with high efficacy and excellent tolerability, approved for use in the treatment of Gram-positive skin and skin structure infections (ABSSSI). Nevertheless, little is known about its pharmacokinetic/pharmacodynamic (PK/PD) properties in real life, which is also due to technical challenges in its quantification in human plasma, preventing an effective application of therapeutic drug monitoring (TDM). In fact, DBV has a high affinity to plasma proteins, possibly resulting in poor recovery after extraction procedure. The aim of this study was to validate a simple, cheap and reliable HPLC-MS method for use in TDM, in accordance with FDA and EMA guidelines. The optimized protein precipitation protocol required 50 μL of plasma, while chromatographic analysis could be performed in 12 min/sample. This method fulfilled the guidelines requirements and then, it was applied for routine DBV TDM in patients receiving off-label high doses (two 1500 + 1500 mg weekly infusions instead of 1000 + 500 mg), with normal renal function or undergoing hemodialysis: continuous hemodiafiltration caused a relevant reduction in DBV exposure, while intermittent dialysis showed comparable DBV concentrations with those of patients with normal renal function. This confirmed the eligibility of the presented method for use in TDM and its usefulness in clinical practice.

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 AUC_0-2w/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.

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.

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.