Validated liquid chromatography–tandem mass spectrometry method for simultaneous quantitation of bendamustine and copanlisib in mouse plasma: Application to a pharmacokinetic study in mice

In this study, we report the development and validation of an LC–tandem mass spectrometry method for the simultaneous quantitation of bendamustine and copanlisib in mouse plasma as per the US FDA regulatory guidelines. The sample processing involves extraction of bendamustine and copanlisib along with internal standard (IS; warfarin) from 50 μL mouse plasma using a liquid–liquid extraction method. The chromatographic separation of bendamustine, copanlisib and the IS was achieved on an Atlantis dC₁₈ column using an isocratic mobile phase (5 mM ammonium acetate:methanol, 20:80 v/v). Bendamustine, copanlisib and the IS eluted at 0.88, 1.39 and 0.74 min, respectively, with a total run time of 2.5 min. The calibration curve ranged from 3.99–2996 and 4.33–3248 ng/mL for bendamustine and copanlisib, respectively. Inter- and intra-day precision and accuracy, stability in processed samples and upon storage, dilution integrity and incurred sample reanalysis were investigated for both the analytes. The intra- and inter-day precisions were in the ranges of 2.01%–5.05% and 2.74%–6.13% and 1.98%–7.64 and 8.62%–9.04% for bendamustine and copanlisib, respectively. Stability studies showed that both analytes were stable on bench top for 6 h, in auto-sampler for 24 and at −80°C for 30 days. The validated method was successfully applied to a pharmacokinetic study in mice.     

Green synthesis and chemical characterization of Thymus vulgaris leaf aqueous extract conjugated gold nanoparticles for the treatment of acute myeloid leukemia in comparison to doxorubicin in a leukemic mouse model

Recently, metallic nanoparticles have been used for the treatment of several disorders, such as cancer. Indeed, finding the chemotherapeutic drug of nanoparticles is in researching the priority of both developed and developing countries. The present study confirms the ability of aqueous extract of Thymus vulgaris grown under in vitro condition for the biosynthesis of gold nanoparticles (AuNPs). Also, in this study, we indicated the antioxidant, cytotoxicity, and anti-acute myeloid leukemia properties of AuNPs compared to doxorubicin in a leukemic mouse model. The synthesized AuNPs were characterized using different techniques including X-ray diffraction (XRD), energy Dispersive X-ray Spectrometry (EDS), fourier-transform infrared spectroscopy (FT-IR) spectroscopy, ultraviolet–visible spectroscopy (UV–Vis.), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). In vivo design, induction of acute myeloid leukemia was done by 7,12-Dimethylbenz[a]anthracene (DMBA) in 75 mice. Then, the animals were randomly divided into six subgroups, including control, untreated, doxorubicin, AuNPs, T. vulgaris, and HAuCl₄. By quantitative real-time PCR, sphingosine-1-phosphate receptor-1 and sphingosine-1-phosphate receptor-5 mRNA expression in lymphocytes were significantly (P ≤ 0.01) raised by treating the leukemic mice with the AuNPs and doxorubicin. Also, AuNPs similar to doxorubicin, significantly (P ≤ 0.01) enhanced the anti-inflammatory cytokines (IL4, IL5, IL10, IL13, and IFNα) and the platelet, lymphocyte, and red blood cell (RBC) parameters and reduced the pro-inflammatory cytokines (IL1, IL6, IL12, IL18, IFNY, and TNFα), and the total white blood cell (WBC), blast, monocyte, neutrophil, eosinophil, and basophil counts as compared to the untreated mice. In vitro design, 2,2-diphenyl-1-picrylhydrazyl (DPPH) test revealed similar antioxidant potentials for doxorubicin and AuNPs. Furthermore, AuNPs similar to doxorubicin had low cell viability dose-dependently against 32D-FLT3-ITD, Human HL-60/vcr, and Murine C1498 cell lines without any cytotoxicity on HUVEC cell line. Above results confirm the excellent antioxidant, cytotoxicity, and anti-acute myeloid leukemia effects of AuNPs compared to doxorubicin. After confirming these results in clinical trial studies, AuNPs can be used as a chemotherapeutic drug for the treatment of acute myeloid leukemia in human.     

Novel green synthesis of Boswellia serrata leaf aqueous extract conjugated gold nanoparticles with excellent anti-acute myeloid leukemia property in comparison to mitoxantrone in a leukemic mice model: Introducing a new chemotherapeutic drug

The present research confirms the capacity of aqueous extract of Boswellia serrata grown under in vitro condition for the green synthesis of gold nanoparticles (AuNPs). Also, we showed the cytotoxicity, antioxidant, and anti-acute myeloid leukemia properties of AuNPs compared to mitoxantrone in a leukemic mouse model. The synthesized AuNPs were characterized using several techniques including XRD, TEM, FE-SEM, UV–Vis, and FT-IR. From the XRD pattern, four distinct diffraction peaks at 38.2°, 44.2°, 64.7° and 77.4° are indexed as (111), (200), (220) and (311) planes of FCC metallic gold. TEM and FE-SEM images revealed an average diameters of 15–30 nm for the nanoparticles. FT-IR findings offered antioxidant compounds in the nanoparticles were the sources of reducing power, reducing gold ions to AuNPs. UV–Vis revealed an absorption band at 536 nm that is related to the surface plasmon resonance of AuNPs. In vivo design, induction of acute myeloid leukemia was done by DMBA in 75 mice. Then, the mice were randomly divided into six subgroups, including untreated, control, HAuCl₄, B. serrata, AuNPs, and mitoxantrone. AuNPs (In the dose of 1 mg/kg body weight) similar to mitoxantrone, significantly (p ≤ 0.05) increased the platelet, lymphocyte, and RBC parameters and the anti-inflammatory cytokines (IL4, IL5, IL10, IL13, and IFNα) and reduced the weights and volumes of liver and spleen and their sub-compartment, the total WBC, blast, monocyte, neutrophil, eosinophil, and basophil counts, and the pro-inflammatory cytokines (IL1, IL6, IL12, IL18, IFNY, and TNFα) as compared to the untreated mice. By quantitative Real-Time PCR, S1PR1 and S1PR5 mRNA expression in lymphocytes were significantly (p ≤ 0.05) increased by treating the leukemic mice with the AuNPs and mitoxantrone. In vitro design, AuNPs similar to mitoxantrone had low cell viability dose-dependently against Human HL-60/vcr, 32D-FLT3-ITD, and Murine C1498 cell lines without any cytotoxicity on HUVEC cell line. Besides, the DPPH assay showed similar antioxidant potentials for AuNPs and mitoxantrone. In conclusion, the results of this research indicated the excellent capacity of synthesized gold nanoparticles using B. serrata leaf aqueous extract in the treatment of acute myeloid leukemia in leukemic mice.     

Modeling mechanical properties of polyhydroxyalkanoate during degradation in animal tissue

Polyhydroxyalkanoates (PHAs) are a family of biodegradable and biocompatible polymers produced by several species microorganisms that possess favorable mechanical properties (e.g. strength and elongation properties). Different types of PHA polymers have been used in medical applications. However, in order to better understand the use of this polymer in the different applications, a thorough understanding of the kinetics of in vivo degradation is one of the major requirements. In this study, poly(3‐hydroxybutyrate) (PHB) was subcutaneously implanted in mice and incubated for 2, 4, 8, or 16 weeks. After removal from the animal, the strength, elongation, mass loss, and enthalpy of the PHB were tested for each time point. From these data, a mathematical model was generated by Rayleigh's method of dimensional analysis, where polymer strength over tissue contact time could be predicted. To prove the model, previous data obtained by our group were used: poly(3‐hydroxybutyrate‐co‐3‐hydroxyhexanoate) [P(HB‐co‐HHx)] incubation in the presence of human embryonic kidney cells (HEK). It was found that the developed model was aligned with experimental results, could predict the strength of the polymer when in contact with cells, and the predicted strength follows the trend of the experimental data. Also, the dimensionless constant (K) value associated with the model is different for both experiments, where this constant, produced via experimental data, is used for construction of a homogeneous equation. Copyright © 2017 John Wiley & Sons, Ltd.     

Development and full validation of an HPLC methodology to quantify atorvastatin and curcumin after their intranasal co‐delivery to mice

There is increasing interest in atorvastatin and curcumin owing to their potential anticancer activity. A new, accurate and sensitive HPLC method was developed, for the first time, to simultaneously quantify atorvastatin and curcumin in mouse plasma and brain, liver, lung and spleen tissues following protein precipitation sample preparation. The chromatographic separation was achieved in 13 min on a C₁₈ column, at 35°C, using a mobile phase composed of acetonitrile–methanol–2% (v/v) acetic acid (37.5:2.5:60, v/v/v) at a flow rate of 1.0 mL/min. The detection of analytes and internal standard was carried out at 247, 425 and 250 nm, respectively. According to international guidelines, the method was shown to be selective, with lower limits of quantification ranging from 10 to 500 ng/mL for curcumin, and from 100 to 600 ng/mL for atorvastatin, linear over a wide concentration range (r² ≥ 0.9971) and with acceptable accuracy (bias ± 12.29%) and precision (coefficient of variation ≤13.15%). The analytes were reproducibly recovered at a percentage >81.10% and demonstrated to be stable under various experimental conditions in all biological matrices. This method can be easily applied to in vivo biodistribution studies related to the intranasal administration of atorvastatin and curcumin, separately or simultaneously.     

Validated HPLC method for simultaneous quantification of mutant IDH1/2 inhibitors (enasidenib,ivosidenib and vorasidenib) in mouse plasma: Application to a pharmacokinetic study

Isocitrate dehydrogenase (IDH) inhibitors comprise a novel class of anticancer drugs, which are approved to treat acute myeloid leukemia patients having mutations on IDH1/2. We report the development and validation of a high‐performance liquid chromatography (HPLC) method for the simultaneous quantitation of IDH inhibitors, namely enasidenib (EDB), ivosidenib (IDB) and vorasidenib (VDB), in mouse plasma as per the US Food and Drug Administration regulatory guidelines. The method involves extraction of EDB, IDB and VDB along with internal standard (IS; phenacetin) from mouse plasma (100 μl) using a simple protein precipitation process. The chromatographic analysis was performed on an HPLC system using a gradient mobile phase (comprising 10 mm ammonium acetate and acetonitrile in a flow‐gradient) and an X‐Terra Phenyl column. The UV detection wave length was set at λ_max 265 nm. EDB, IDB, VDB and the IS eluted at 7.36, 8.60, 9.50 and 5.12 min, respectively, with a total run time of 10 min. The calibration curve was linear over a concentration range of 0.20–12.5 μg/ml for EDB and 0.50–12.5 μg/ml for IDB and VDB (r² = ≥0.998 for all of the analytes). Validation results met the acceptance criteria. The validated HPLC method was successfully applied to a pharmacokinetic study in mice.     

A dried blood spot assay with HPLC–MS/MS for the determination of larotrectinib in mouse blood and its application to a pharmacokinetic study

Larotrectinib is a first-generation tropomyosin kinase inhibitor, approved for the treatment of solid tumors. In this paper, we present a validated dried blood spot (DBS) method for the quantitation of larotrectinib from mouse blood using HPLC–MS/MS, which was operated under multiple reaction monitoring mode. To the DBS disc cards, acidified methanol enriched with internal standard (IS; enasidenib) was added and extracted using tert-butyl methyl ether as an extraction solvent with sonication. Chromatographic separation of larotrectinib and the IS was achieved on an Atlantis dC₁₈ column using 10 mm ammonium formate–acetonitrile (30:70, v/v) delivered at a flow-rate of 0.80 ml/min. Under these optimized conditions, the retention times of larotrectinib and the IS were ~0.93 and 1.37 min, respectively. The total run time was 2.50 min. Larotrectinib and the IS were analyzed using positive ion scan mode and parent–daughter mass to charge ion (m/z) transitions of 429.1 → 342.1 and 474.1 → 267.1, respectively, were used for the quantitation. The calibration range was 1.06–5,080 ng/ml. No matrix effect or carryover was observed. Hematocrit did not influence DBS larotrectinib concentrations. All of the validation parameters met the acceptance criteria. The applicability of the validated method was shown in a mouse pharmacokinetic study.     

Quantitative analysis of enasidenib in dried blood spots of mouse blood using an increased‐sensitivity LC–MS/MS method: Application to a pharmacokinetic study

A simple, sensitive and rapid assay method has been developed and validated as per regulatory guidelines for the estimation of enasidenib on mouse dried blood spots (DBS) using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. The method employs liquid extraction of enasidenib from DBS disks of mouse whole blood followed by chromatographic separation using 0.2% formic acid–acetonitrile (25:75, v/v) at a flow rate of 1.0 mL/min on an Atlantis dC₁₈ column with a total run time of 2.0 min. The MS/MS ion transitions monitored were m/z 474.0 → 267.1 for enasidenib and m/z 309.2 → 251.3 for the internal standard (warfarin). The assay was linear in the range of 1.01 – 3044 ng/mL. The within‐run and between‐run precisions were in the range of 3.18 – 9.06 and 4.66 – 8.69%, respectively. Stability studies showed that enasidenib was stable on DBS cards for 1 month. This novel method has been applied to analyze the DBS samples of enasidenib obtained from a pharmacokinetic study in mice.     

Pharmacokinetics and metabolism study of veratramine in mice after oral administration using LC‐MS/MS

A simple and sensitive high‐performance liquid chromatography coupled with hybrid triple quadrupole–linear ion trap mass spectrometry (Q‐trap‐MS) method was developed and validated for the determination of veratramine, the major bioactive and neurotoxic component in Veratrum nigrum L. Veratramine and the internal standard (IS) were separated with a Waters Symmetry C₁₈ column and eluted with a gradient mobile phase system containing acetonitrile and 0.1% aqueous formic acid. The analysis was performed by using positive electrospray ionization mode with multiple reaction monitoring (MRM). Transition ions of m/z 410.2 → 295.2 for veratramine and m/z 426.1 → 113.8 for the IS were monitored. The method was validated with a good linearity in the range of 1–1000 ng/mL and lower limit of quantification of 1 ng/mL. The precision (CV) of intra‐ and inter‐day ranged from 3.92 to 7.29%, while the accuracy (bias) intra‐ and inter‐day were between ?4.78 and 1.65%. The recovery, stability and matrix effect were within the acceptable ranges. Five metabolites of veratramine, including four hydroxylated and one sulfated metabolites, were tentatively identified using predictive MRM–information dependent acquisition–enhanced product ion mode (predictive MRM‐IDA‐EPI). The developed method was successfully applied to the pharmacokinetic and metabolic study of veratramine in mice after oral administration of veratramine. Copyright © 2016 John Wiley & Sons, Ltd.     

Effect of Sulfation Route and Subsequent Oxidation on Derivatization Degree and Biocompatibility of Cellulose Sulfates

Sulfated cellulose (CS) represents an interesting biopolymer due to bioactivity comparable to heparin. However, use of CS for making surface coatings or hydrogels requires the presence of reactive groups for covalent reactions. Here, an approach is presented to oxidize cellulose sulfates for subsequent cross‐linking reactions with amino groups to form imine bonds. Cellulose is sulfated by direct sulfation or acetosulfation, followed by a M alaprade oxidation. The CS obtained is characterized by elemental analysis and ¹³C‐NMR spectroscopy. The resulting oxidized cellulose sulfates (oxCS) have different degrees of sulfation ranging from 0.79 to 1.13 and oxidation degrees from 0.18 to 0.34, but also different mass average molecular mass (M_W). Toxicity studies are carried out with mouse 3T3 fibroblasts exposed to aqueous solutions of oxCS. The results show that all oxCS are non‐toxic at lower concentrations (0.5 mg mL^?1), but with both increasing degree of oxidation and concentrations, toxic effects are observed particularly for acetosulfated and lesser for direct sulfated oxCS, which is related to a decrease in the M_W of the products. It is concluded that oxCS obtained by direct sulfation with M_W above 70 kDa may represent a biocompatible material for the applications suggested above.