Pilot Pharmacokinetic Study Research Articles

A pilot pharmacokinetic and Metabolite identification study of Erinacine A in a Single landrace pig model

Erinacine A has been proven to have the ability to protect nerves and have the benefit of neurohealth. However, the pharmacokinetic and metabolites study of erinacine A in pigs, whose physiology and anatomy are similar to humans, have not been reported. In this study, 5 mg/kg of erinacine A was intravenously administered to the landrace pig. Blood, cerebrospinal fluid, and brain tissue samples were collected and analyzed by HPLC-QQQ/MS and UPLC-QTOF/MS. The results indicated the following pharmacokinetic parameters in plasma samples: with an area under the plasma concentration versus time curve (AUC) were 38.02 ± 0.03 mg∙min/L (AUC0-60) and 43.60 ± 0.06 mg∙min/L (AUC0-∞), clearance (CL) was 0.11 ± 0.00 L/min∙kg, volume of distribution (Vd) was 4.24 ± 0.00 L/kg, and terminal half-life (T1/2β) was 20.85 ± 0.03 min. In the cerebrospinal fluid samples, erinacine A was detected after 15 min and the highest concentration (5.26 ± 0.58 μg/L) was observed at 30 min. In the brain tissue sample, 77.45 ± 0.58 μg/L of erinacine A was found. In the study of metabolites, there were 6 identical metabolites in plasma and brain tissue. To our surprise, erinacine B was found to be the metabolite of erinacine A, and its concentration increased over time as erinacine A was metabolized. In summary, this study is the first to demonstrate that erinacine A can be found in the cerebrospinal fluid of landrace pigs. Additionally, the metabolite identification of erinacine A in landrace pigs is also investigated.

Validation of a bioanalytical HPLC-UV method to quantify Α-Bisabolol in rat plasma applied to pharmacokinetic pilot study with the drug nanoemulsion.

α-Bisabolol (α-BIS) is a sesquiterpene alcohol present in chamomile essential oil [Chamomilla recutita (L.) Rauschert]. Despite its numerous pharmacological effects, its pharmacokinetics remain understudied. An analytical method capable of quantifying α-BIS in plasma is crucial to enable pharmacokinetic analysis. Presently, only one study has quantified it using mass spectrometry. Administering α-BIS requires a nanoemulsion for intravenous injection. This study aimed to develop and validate a bioanalytical method using high-performance liquid chromatography with an ultraviolet detector to quantify α-BIS in rat plasma. The method employed acetonitrile and ultrapure water (80:20, v/v) as the mobile phase, with a flow rate of 1ml/min and concentrations ranging from 465 to 29.625 μg/ml. All US Food and Drug Administration-designated assays were successful, indicating the method's precision, accuracy, sensitivity and linearity in determining α-BIS in rat plasma. The developed nanoemulsion, assessed through dynamic light scattering analysis, the ensemble collection of particles and polydispersity index evaluation, proved safe and effective for intravenous administration. The pharmacokinetic parameters such as volume of distribution, clearance and half-life indicated that α-BIS tends to persist in the body. This study provides a foundation for further research to explore α-BIS's potential pharmaceutical applications in the future.

Simultaneous quantification and confirmation of oxycodone and its metabolites in equine urine using ultra-high performance liquid chromatography-tandem mass spectrometry

Oxycodone, an opioid commonly used to treat pain in humans, has the potential to be abused in racehorses to enhance their performance. To understand the pharmacokinetics of oxycodone and its metabolites in horses, as well as to detect the illegal use of oxycodone in racehorses, a method for quantification and confirmation of oxycodone and its metabolites is needed. In this study, we developed and validated an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method that can simultaneously quantify and confirm oxycodone and eight metabolites in equine urine. Samples were subjected to enzymatic hydrolysis and then liquid–liquid extraction using ethyl acetate. The analyte separation was achieved on a Hypersil Gold C18 sub-2 µm column and analytes were detected on a triple quadrupole mass spectrometer. The limit of detection (LOD) and lower limit of quantification (LLOQ) were 25–50 pg/mL and 100 pg/mL, respectively. Excellent linearity of the calibration curves was observed over a range of 100–10000 pg/mL for all nine analytes. Retention time, signal-to-noise ratio, and product ion ratios were utilized as confirmation criteria, with the limits of confirmation (LOC) ranging from 100 to 250 pg/mL. The data from a pilot pharmacokinetic (PK) study suggested that oxycodone metabolites have longer detection periods in equine urine compared to oxycodone itself; thus, the detection of metabolites in equine urine extends the ability to detect oxycodone exposure in racehorses.

Distribution of Bevacizumab into the Cerebrospinal Fluid of Children and Adolescents with Recurrent Brain Tumors.

To date, evidence has been lacking regarding bevacizumab pharmacokinetics in the cerebrospinal fluid (CSF). This study assessed the penetration of bevacizumab, as part of a metronomic antiangiogenic treatment regimen, into the CSF of children, adolescents, and young adults with recurrent brain tumors. Serum and CSF concentrations, malignant cells, and vascular endothelial growth factor A (VEGF-A) were analyzed in 12 patients (5-27 years) following 10 mg/kg bevacizumab intravenous biweekly administration (EudraCT number 2009-013024-23). A population pharmacokinetic model including body weight, albumin, and tumor type as influential factors was extended to quantify the CSF penetration of bevacizumab. Apart from in serum (minimum concentration/maximum concentration [Cmin/Cmax] 77.0-305/267-612 mg/L, median 144/417mg/L), bevacizumab could be quantified in the CSF (0.01-2.26mg/L, median 0.35 mg/L). The CSF/serum ratio was 0.16 and highly variable between patients. Malignant cells could be detected in CSF before initiation of treatment in five of 12 patients; after treatment, the CSF was cleared in all patients. VEGF-A was detected in three patients before treatment (mean ± SD: 20 ± 11 pg/mL), and was still measurable in one of these patients despite treatment (16 pg/mL). This pharmacokinetic pilot study indicated penetration of bevacizumab into the CSF in a population of children, adolescents, and young adults with recurrent brain tumors.

Production of antibody and development of enzyme-linked immunosorbent assay for therapeutic drug monitoring of eravacycline

Eravacycline (ERC) was approved for clinical use in 2018. It is more potent than other tetracyclines and can overcome resistance, making it an attractive option for combating multidrug-resistant bacterial infections. Intensive pharmacokinetic (PK) studies are currently being conducted to ensure the effectiveness and safety of ERC in various groups of patients, including those undergoing extracorporeal therapies. This study is the first attempt to develop a simple, efficient, and high-throughput immunoassay for quantifying ERC in human or animal serum. BSA-ERC conjugate as immunogen elicited antibody production in rabbits. Monitoring of the immune response and comparison of homologous and heterologous coating antigens allowed selection of immunoreagents and development of an assay that was selective for ERC possessing sensitivity (IC50), dynamic range (IC20-IC80) and detection limit equal to 3.3 ng/mL, 0.27–54 ng/mL and 0.09 ng/mL, respectively. The developed ELISA showed acceptable recovery of ERC (85–105 %) from rabbit and human serum in the clinically relevant concentration range of 0.1–3.0 mg/L. The method was used to quantify serum ERC concentration in the pilot PK study in Soviet chinchilla rabbits. The results were confirmed by HPLC-MS/MS.

Development and validation of an UPLC-MS/MS method for the determination of irinotecan (CPT-11), SN-38 and SN-38 glucuronide in human plasma and peritoneal tumor tissue from patients with peritoneal carcinomatosis

Irinotecan (CPT-11), an antineoplastic drug, is used for the treatment of colorectal and pancreatic cancer due to its topoisomerase I inhibitory activity. CPT-11 is a prodrug which is converted to its active metabolite SN-38 by carboxylesterases. SN-38 is further metabolized to its inactive metabolite SN-38 glucuronide. When evaluating the pharmacokinetic properties of CPT-11 and its metabolites, it is important to accurately assess the concentrations in both plasma as well as tumor tissues. Therefore, the aim of the current study was to develop and validate a robust and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry method to quantify the concentration of CPT-11 and its metabolites (SN-38 and SN-38 glucuronide) in human plasma and peritoneal tumor tissue. The sample preparation of plasma and tumor tissue consisted of protein precipitation and enzymatic digestion/liquid–liquid extraction, respectively. Chromatographic separation was achieved with an Acquity UPLC BEH C18 column combined with a VanGuard pre-column. The mobile phases consisted of water +0.1 % formic acid (mobile phase A) and acetonitrile +0.1 % formic acid (mobile phase B). Mass analysis was performed using a Xevo TQS tandem mass spectrometer in the positive electrospray ionization mode. Method validation was successfully performed by assessing linearity, precision and accuracy, lower limit of quantification, carry over, selectivity, matrix effect and stability according to the following guidelines: “Committee for Medicinal Products for Human use, Guideline on Bioanalytical Method Validation”. A cross-validation of the developed method was performed in a pilot pharmacokinetic study, demonstrating the usefulness of the current method to quantify CPT-11 and its metabolites in the different matrices.

Validation of a quantitative multiplex LC-MS/MS assay of carvedilol, enalaprilat, and perindoprilat in dried blood spots from heart failure patients and its cross validation with a plasma assay

IntroductionAdherence to medication is an important determinant of outcomes in chronic diseases like heart failure. Drug assays provide objective adherence biomarkers. Dried blood spots (DBS) are appealing samples for drug assays due to less demanding transportation and storage requirements. ObjectivesTo analytically validate a LC-MS/MS method for the simultaneous quantification of carvedilol, enalaprilat, and perindoprilat in DBS and evaluate the feasibility of using the method as an adherence determining assay. To validate the assay further clinically by establishing correlation and agreement between plasma and DBS samples from a pharmacokinetic pilot study. MethodsThe method was validated over a concentration range of 1.00–200 ng/mL according to FDA guidelines. Adherence tracking ability of the assay was evaluated using a pharmacokinetic pilot study. Correlation and agreement were evaluated through Deming regression and Bland-Altman analysis, respectively. ResultsAccuracy, precision, selectivity, and sensitivity were proven with complete and reproducible extraction recovery at all concentrations tested. Stability of the analytes in the matrix and throughout sample processing was proven. The full range of concentrations of the pharmacokinetic pilot study could be quantified for enalaprilat, but not for carvedilol and perindoprilat. The difference between the observed and calculated plasma concentrations was less than 20 % of their mean for >67 % of samples for all analytes. ConclusionsThe assay is suitable as a screening tool for carvedilol and perindoprilat, while suitable as an adherence determining assay for enalaprilat. Equivalence between observed and predicted plasma concentrations proves DBS and plasma concentrations can be used interchangeably.

Pilot Pharmacokinetic Study in Healthy Adults Using Intravascular Microdialysis Catheters Modified for Use in Paediatric Patients to Assess Vancomycin Blood Levels.

Exhaustive pharmacokinetic (PK) studies in paediatric patients are unavailable for most antibiotics and feasibility of PK studies is limited by challenges, such as low blood volume and venipuncture-related pain. Microdialysis (MD) represents a promising method to overcome these obstacles. The aim of this proof-of-concept study was to develop and validate modified MD catheters that can be used to obtain concentration-time profiles of antibiotics in paediatric patients. Following extensive in vitro MD experiments, a prospective open-labelled study in ten healthy adult volunteers (HVs) was conducted. Subjects received a single intravenous dose of 1000 mg vancomycin, then plasma and intravascular microdialysate were sampled over 24 h. In vivo MD probe calibration was conducted using the retrodialysis technique. Plasma protein binding was measured using ultrafiltration. Confirmation of the measurements was performed using a Bland-Altman plot, relevant PK parameters were calculated, and a pharmacometric model was established. No safety issues were encountered. The concentration-time curves of microdialysate and plasma measurements showed good alignment. The Bland-Altman plot yielded a mean bias of 0.19 mg/L and 95% limits of agreement of -9.34 to 9.71 mg/L. A two-compartment model best described plasma PK, model-based estimates for recovery of the MD probes being in high agreement with the observed values. Quantified estimates of fraction unbound were comparable between plasma and microdialysate (p = 0.56). An innovative MD catheter that can be inserted into small intravenous lines was successfully developed and applied in HV. This proof-of-concept study is encouraging and opens the way to further experiments leading towards future use of MD in paediatric patients.

Fast determination of free metronidazole in rat plasma and peritoneal fluid using HPLC-UV method.

Peritonitis refers to the inflammatory reaction of the peritoneum to aggression. In addition, it contributes significantly to sepsis. The presence of free concentrations of antimicrobials above the minimum inhibitory concentration at the site of infection is critical to therapeutic response. Metronidazole (MTZ) is an antimicrobial used to treat peritonitis because of its effectiveness against anaerobic microorganisms. This study investigates free MTZ concentrations in peritoneal microdialysate in Wistar rats. A C18 column (150 × 4.0mm, 5μm) was used for the analysis conducted at 40°C under isocratic conditions. The mobile phase consisted of acetonitrile and an aqueous solution of 50-mM monobasic phosphate buffer and 0.1% triethylamine, with pH3.0 (10:90, v/v). MTZ calibration was linear in the range of 0.5-30.0μg/ml. The intra- and inter-day precision was satisfactory with relative standard deviation ≤5.67%. The accuracy ranged from 90.64 to 103.77%, and the lower limit of quantification was 0.5μg/ml. The developed method was successfully applied in a pilot pharmacokinetic study after MTZ administration (30 mg/kg, intravenously) in rats. The main advantage of the employed method is that it does not require sample processing and protein removal steps. This is the first study to be conducted using MTZ in rats.

Fixed Dose Combination Tablets of Aripiprazole and Divalproex Sodium: a Pilot Pharmacokinetic Study in Human Volunteers.

A combination product of aripiprazole (antipsychotic) and divalproex sodium (mood stabilizer) was recently developed to establish their tolerability and safety in fixed dose combination (FDC). A pilot pharmacokinetic (PK) open-labeled parallel study on healthy human volunteers was carried out to assess the PK of FDC of aripiprazole/divalproex sodium in comparison with its individual components with a view to rationalize therapeutic regimen and potentially improve compliance of bipolar patients in future. A total of 24 volunteers were randomized to aripiprazole 5mg, divalproex sodium 500mg, and FDC (aripiprazole/divalproex sodium 5/500 mg) enteric-coated tablets. Peak plasma concentration (Cmax) and time to reach peak plasma concentration (Tmax) of aripiprazole increased, Cmax of valproic acid increased, and Tmax decreased. Half-life (t1/2) of both aripiprazole and valproic acid decreased. Area under the curve (AUC) of both aripiprazole and valproic acid increased while volume of distribution (Vd) and clearance (Cl) decreased when used in fixed combination. Increase in the AUC and decrease in the Vd of aripiprazole in the presence of valproic acid were found statistically significant while rest of the parameters were insignificant at level 0.05. Although not adequately powered, this pilot study gives an idea that FDC of aripiprazole and divalproex sodium has a PK profile comparable to its mono-component products. Thus, concomitant use of aripiprazole and valproate in FDC is possible which may prove to be a cost-effective and result-oriented substitute for conventional individual tablets to improve patients' compliance; however, further evaluation with positive control is required.

Development and validation of an LC-ESI-QTOF-MS method to measure cefepime in the plasma and peritoneal fluid of rats using microdialysis: Application in a pilot pharmacokinetic study.

Cefepime (CEF) is a cephalosporin and can be administered in secondary peritonitis together with metronidazole to treat sepsis. This study aimed to develop and validate an LC-ESI-QTOF-MS method for the quantification of cefepime in the plasma and peritoneal microdialysate of healthy Wistar rats. Chromatographic separation was performed using a CLC-ODS C18 column (250 ×4.6mm), a C18 pre-column (4mm, 5μm) and isocratic elution. Gallic acid was used as the internal standard. The mobile phase consisted of (A) ultrapure water (pH adjusted to 3.5) and (B) acetonitrile (80:20, v/v) at 0.8ml/min. Quantification was performed using a mass spectrometer with electrospray ionization in positive mode to monitor ions with m/z 481.1322 (CEF) and m/z 171.0288 (IS). The method was validated for selectivity, precision, accuracy, linearity, stability, lower limit of quantification, carryover, recovery and matrix effect. Calibration was done in the ranges 1-40 and 1-100 μg/ml for the peritoneal microdialysate and plasma, respectively. Plasma extraction recovery ranged from 93.9 to 99.9%. The technique was validated and successfully applied in a pilot pharmacokinetic study for estimating the free concentration of CEF in the peritoneal microdialysate of rats for the first time.

Determination of plasma and tissue distribution of 27-hydroxycholesterol after a single oral administration in a mouse model

Background The side chain 27-hydroxycholesterol has been reported to inhibit the replication of several pathogen viruses, including herpes simplex virus, rhinovirus, rotavirus and SARS-CoV-2, in in vitro and ex vivo models. Objective In view of a future potential therapeutic use of 27-hydroxycholesterol, a pilot pharmacokinetic study was set up. Methods This active substance was complexed with 2-hydroxypropyl-β-cyclodextrin and orally administered in a single dose to CD1 male mice; its recovery in plasma and a few tissues up to 24 h post-treatment was evaluated. Results The absorption of the oxysterol by the small intestine was moderate, due to its physicochemical properties, but still relevant and rapid, showing a peak at 1 h after supplementation and being almost completed 24 h after treatment. 27-Hydroxycholesterol appeared to be a high hepatic extraction drug, possibly with an extrahepatic component contributing to the total clearance. Conclusions Following the oral 25 mg/kg dosing, plasma levels of 27-hydroxycholesterol showed an average steady-state concentration similar to that shown to be able to inhibit the replication of all viruses tested so far in in vitro models. Significance statement The first pharmacokinetic data relative to a natural oxysterol administered p.o. are reported. Data should contribute to further elucidate oxysterol pathophysiology and guide non-clinical studies aiming at investigating possible therapeutic use of 27-hydroxycholesterol or its analogs.

Therapeutic Drug Monitoring of Mycophenolic Acid as a Precision Medicine Tool for Heart Transplant Patients: Results of an Observational Pharmacokinetic Pilot Study.

In the clinical practice management of heart transplant (HTx), the impact of calcineurin inhibitors co-administration on pharmacokinetics (PKs) of mycophenolic acid (MPA), mycophenolate mofetil (MMF) active drug, is not adequately considered. This retrospective study investigated full MPA-PK profiles by therapeutic drug monitoring (TDM) in 21 HTx recipients treated with MMF combined with cyclosporine (CsA) or tacrolimus (TAC) at a median time of 2.6 months post-transplant. The two treatment groups were compared. We described the main MPA-PK parameters in patients developing acute cellular rejection (ACR) and those who did not. Median dose-adjusted MPA-trough levels and MPA-AUC0–12h were higher in patients co-treated with TAC than with CsA (p = 0.0001 and p = 0.006, respectively). MPA-Cmax and Tmax were similar between the two groups, whereas the enterohepatic recirculation biomarker of MPA (MPA-AUC4–12h) was higher in the MMF and TAC group (p = 0.004). Consistently, MPA clearance was higher in the MMF and CsA group (p = 0.006). In total, 87.5% of ACR patients were treated with MMF and CsA, presenting a lower MPA-AUC0–12h (p = 0.02). This real-world study suggested the CsA interference on MPA-PK in HTx, evidencing the pivotal role of MPA TDM as a precision medicine tool in the early phase after HTx. A prospective study is mandatory to investigate this approach to HTx clinical outcomes.

Simultaneous Determination of Carvedilol, Enalaprilat, and Perindoprilat in Human Plasma Using LC\u2013MS/MS and Its Application to a Pharmacokinetic Pilot Study

A method for the extraction and quantification of carvedilol, enalaprilat, and perindoprilat in 50 µL human plasma, using high-performance liquid chromatography with tandem mass spectrometry (LC–MS/MS) detection was developed and validated. Samples were prepared via protein precipitation with chromatographic separation on a Restek Ultra II Biphenyl column using gradient elution at a corresponding flowrate of 300 µL/min. Electrospray ionisation with mass detection at unit resolution in the multiple reaction monitoring (MRM) mode on an AB Sciex API 5500 mass spectrometer was used. Accuracy, precision, selectivity, sensitivity, matrix effects, recovery, process efficiency, and stability were assessed over the validation period. The assay was validated over the calibration range 0.2–200 ng/mL for all three analytes. The inter- and intra-day precision expressed as the coefficient of variation (CV) and accuracy (%Nom) all fell within acceptable limits. The overall recovery was calculated as 72.9%, 77.1%, and 77.0% for carvedilol, enalaprilat, and perindoprilat respectively, with the recovery being shown to be reproducible at the low, medium and high end of the calibration range for all three analytes. The method proved to be specific for all three analytes with no significant matrix effects observed. The validated method facilitated the analysis of carvedilol, enalaprilat, and perindoprilat in human plasma collected from adults as part of a pilot pharmacokinetic study. This validated analytical method lays the foundation for determining adherence in heart failure patients prescribed with carvedilol, enalapril and perindopril.

Calculating the Charcoal Blockade Efficiency for Bioequivalence Study of Inhaled Ipratropium Bromide Using A Model Method

Charcoal blockade is a useful approach to block gastrointestinal (GI) absorption of orally inhaled drug products (OIDPs) and therefore can be used effectively to determine drug absorption exclusively via the pulmonary route. Charcoal blockade efficiency (CBE) should be measured to show whether adequate blockade of GI exposure is achieved in bioequivalence (BE) study. The purpose of this study is to employ a model method to calculate the CBE for a pilot pharmacokinetic (PK) BE study of inhaled ipratropium bromide. This model method, based on a convolution integral, is built in-house using MATLAB package. The results demonstrated a full blockade of GI absorption of ipratropium bromide for both test and reference drug products. This study has shown that the model method may provide a useful approach for validation of charcoal blockade method used in PK BE study for OIDPs. The ability to use modeling may simplify human PK studies in general, and is particularly valuable when for ethical, technical or regulatory reasons administration of an orally swallowed form of the drug is not possible.

Part II : Statistics in practice: Study design and application of inferential statistics—Interventional research

Part <scp>II</scp> : Statistics in practice: Study design and application of inferential statistics—Interventional research

A validated LC-MS/MS method for the quantitation of cefazolin in human adipose tissue: Application of EMR-Lipid sorbent as an efficient sample clean-up before mass spectrometric analyses

A novel, simple, rapid, and sensitive high-performance liquid chromatography-tandem mass spectrometry method was developed to determine cefazolin concentrations in human adipose tissue. Sample preparation was performed by protein precipitation followed by using Captiva EMR-Lipid plates. The mobile phase consisted of 5 mM ammonium formate and 0.1% formic acid in water and 0.1% formic acid in ACN, and was pumped through a Synergi Fusion-RP column with a gradient elution program at a flow rate of 0.3 mL/min. The mass spectrometer was operated in a positive ion mode. Cloxacillin was used as an internal standard due to the observed cross-signal contribution between cefazolin and 13C2,15N-cefazolin. The method was validated according to the FDA and EMA guidelines and passed all the acceptance criteria. The calibration range was 0.05–50 µg/mL in adipose tissue homogenate (0.15–150 µg/g in adipose tissue), precision CV < 4.5%, accuracy within 93.1–100.4%. The carry-over was negligible, recovery of the method was high, and no significant matrix effect was present. Rat subcutaneous adipose tissue was demonstrated to be a suitable surrogate matrix for human adipose tissue. The validated method was successfully applied in a pilot pharmacokinetic study and will further be used in a large cohort of non-obese and obese patients dosed prophylactically with cefazolin before surgeries.

Monthly Increase in Vitamin D Levels upon Supplementation with 2000 IU/Day in Healthy Volunteers: Result from "Integriamoci", a Pilot Pharmacokinetic Study.

Vitamin D (VD) is a calcium- and phosphate-controlling hormone used to treat bone disorders; yet, several other effects are progressively emerging. VD deficiency is highly prevalent worldwide, with suboptimal exposure to sunlight listed among the leading causes: oral supplementation with either cholecalciferol or calcitriol is used. However, there is a scarcity of clinical studies investigating how quickly VD concentrations can increase after supplementation. In this pilot study, the commercial supplement ImmuD3 (by Erboristeria Magentina®) was chosen as the source of VD and 2000 IU/day was administered for one month to 21 healthy volunteers that had not taken any other VD supplements in the previous 30 days. Plasma VD levels were measured through liquid chromatography coupled to tandem mass spectrometry after 7, 14, and 28 days of supplementation. We found that 95% of the participants had insufficient VD levels at baseline (<30 ng/mL; median 23.72 ng/mL; IQR 18.10–26.15), but after 28 days of supplementation, this percentage dropped to 62% (median 28.35 ng/mL; IQR 25.78–35.20). The median increase in VD level was 3.09 ng/mL (IQR 1.60–5.68) after 7 days and 8.85 ng/mL (IQR 2.85–13.97F) after 28 days. This study suggests the need for continuing VD supplementation and for measuring target level attainment.

Curcumin Loaded PEGylated Nanoemulsions Designed for Maintained Antioxidant Effects and Improved Bioavailability: A Pilot Study on Rats.

The current study describes the experimental design guided development of PEGylated nanoemulsions as parenteral delivery systems for curcumin, a powerful antioxidant, as well as the evaluation of their physicochemical characteristics and antioxidant activity during the two years of storage. Experimental design setup helped development of nanoemulsion templates with critical quality attributes in line with parenteral application route. Curcumin-loaded nanoemulsions showed mean droplet size about 105 nm, polydispersity index <0.15, zeta potential of −40 mV, and acceptable osmolality of about 550 mOsm/kg. After two years of storage at room temperature, all formulations remained stable. Moreover, antioxidant activity remained intact, as demonstrated by DPPH (IC50 values 0.078–0.075 mg/mL after two years) and FRAPS assays. In vitro release testing proved that PEGylated phospholipids slowed down the curcumin release from nanoemulsions. The nanoemulsion carrier has been proven safe by the MTT test conducted with MRC-5 cell line, and effective on LS cell line. Results from the pharmacokinetic pilot study implied the PEGylated nanoemulsions improved plasma residence of curcumin 20 min after intravenous administration, compared to the non-PEGylated nanoemulsion (two-fold higher) or curcumin solution (three-fold higher). Overall, conclusion suggests that developed PEGylated nanoemulsions present an acceptable delivery system for parenteral administration of curcumin, being effective in preserving its stability and antioxidant capacity at the level highly comparable to the initial findings.

Eco-friendly UPLC-MS/MS analysis of possible add-on therapy for COVID-19 in human plasma: Insights of greenness assessment

Facing the pandemic COVID-19 is of highest priority for all researchers nowadays. Recent statistics indicate that the majority of the cases are home-treated. Two drugs of interest, Guaifenesin and Bromohexine HCl, are among the add-on therapy for treatment of COVID-19 mild cases, which has raised the need for their simultaneous determination. The analysis of the two drugs of interest was described using ultra-performance liquid chromatography–tandem mass spectrometric (UPLC–MS/MS) in plasma of healthy human volunteers using tetryzoline HCl as an internal standard (IS) after liquid–liquid extraction. The applied chromatographic conditions were Kinetex C18 (100 Å, 2.6 µm X 50 mm X 4.6 mm) column and a mixture of methanol: water (95: 5, v/v) as a mobile phase at flow rate 1 mL/min. The positive ionization mode was used for detecting the ions, by observing the pairs of transition m/z 199 < 125 for GUF, m/z 377 < 114 for BRM and m/z 201 < 131 for IS. The linearity range was from 50 to 1500 ng/mL for GUF and 0.5–50 µg/mL for BRM. Limit of detection (LOD) was found to be 35.16 and 0.43 ng/ml for GUF and BRM, respectively. The method was validated according to FDA guidance. The proposed method was assessed to be more eco-friendly versus the reported method using the greenness assessment tools: National Environmental Methods Index (NEMI), Assessment of Green Profile (AGP), Green Analytical Procedure Index (GAPI) and Eco-Scale. The proposed method was applied for the application of a pilot pharmacokinetic study.