Concentrations Of Daunorubicin Research Articles

Investigation of the Cytotoxic and Antiproliferative Effects of Liposomal Daunorubicin on Human Colorectal Cancer (HCT116) Cell Line

Background: The incidence of colorectal cancer is increasing globally. Daunorubicin (DNR), an anthracycline antibiotic, is effective against various cancers. The PI3K/AKT/mTOR signaling pathway is crucial in regulating cell growth and cancer growth. Objectives: This study aims to evaluate the effects of liposomal daunorubicin (Lip-DNR) on cell proliferation and cell death induction in HCT116 cells compared to free daunorubicin. Methods: Lip-DNR was synthesized, and its shape and size were analyzed using FE-SEM imaging. HCT116 cells were treated with Lip-DNR concentrations of 0 (control), 0.125, 0.25, 0.5, 1, and 2 μM for 48 hours to determine the IC50. The effects of free (0.5 μM) and liposomal DNR (IC50 of 0.43 μM) on PI3K mRNA levels were assessed using real-time PCR. The cell cycle was analyzed by flow cytometry. Results: FE-SEM imaging showed that the liposomes are spherical and range from 50 - 100 nm in size. Lip-DNR induced cell death in HCT116 cells in a dose-dependent manner, with 0.5 μM Lip-DNR causing more cell death than an equivalent concentration of free DNR. Analysis of PI3K gene expression showed that DNR decreases PI3K gene transcription in HCT116 cells, with Lip-DNR having a more substantial effect than the free form. Both forms reduced the proportion of G2/M phase cells, but Lip-DNR was more effective at inhibiting cell proliferation in HCT116 cells. Conclusions: DNR inhibits the proliferation of HCT116 cells by downregulating PI3K gene expression and enhancing cell death, with the liposomal form demonstrating stronger effects than the free form.

Sensitive determination of daunorubicin in plasma of children with leukemia using pH-switchable deep eutectic solvents and HPLC-UV analysis

An environmental friendly, fast, easy and inexpensive liquid-liquid microextraction (LLME) in combination with pH-switchable deep eutectic solvent (DES) method followed by HPLC was investigated for the separation and determination of daunorubicin (DNR) in human plasma samples. For this purpose, first, 9 DESs were prepared based on previous studies and their switchability in aqueous solution was evaluated by changing the pH. Non-switchable DESs were discarded and switchable DESs were used to extract DNR. The parameters affecting the extraction efficiency were optimized (DES type, volume of DES, concentration of KOH, volume of HCl, salt addition and extraction time). After optimizing the conditions and drawing the calibration curve, figures of merit were calculated. Relative standard deviations (%RSDs) based on 7 replicate with 50 μg L−1 of DNR in plasma were 2.7 for intra-day and 4.8 % for inter-day. A wide linear range from 0.15 to 200 μg L−1 was obtained. The detection limit of the method based on signal-to-noise 3 and the quantification limit of the method based on signal-to-noise 10 were 0.05 and 0.15, respectively. After spiking plasma samples with different concentrations of DNR, relative recoveries were obtained in the range of 91.0–107.8 %.

Basic Research on Therapy of Leukemia with Nanoparticles Drug Delivery System Modified by Transferrin Receptor Monoclonal Antibody

To investigate the therapeutic effect of targeted drug-loaded nanoparticles modified by transferrin receptor monoclonal antibody (TfR mAb) on acute leukemia and its potential anti-tumor mechanism. Nanoparticles drug delivery system, which was composed of poly (lactic-co-glycolic acid), poly-l-lysine, polyethylene glycol, TfR mAb (TfR mAb-PLGA-PLL-PEG)-daunorubicin (DNR), was first synthesized. After drug intervention, the intracellular accumulation in leukemia HL60 cells was observed under a fluorescent microscope and concentration of DNR was determined by flow cytometry (FCM). Meanwhile, cell apoptosis rate was measured by FCM and the expression levels of apoptosis related protein Cleaved-caspase 3 was determined by Western blot. Under an inverted fluorescent microscope, intracellular accumulation of DNR autofluorescence in HL60 cells was observed in both TfR mAb-PLGA-PLL-PEG-DNR group and DNR group. FCM analysis showed that the intracellular concentration of DNR in TfR mAb-PLGA-PLL-PEG-DNR group was higher than that in DNR group（P<0.05）. The apoptotic rate of HL60 cells in TfR mAb-PLGA-PLL-PEG-DNR group was higher than that of DNR group（P<0.05）. Moreover, the expression levels of apoptosis-related protein Cleaved-caspase 3 in TfR mAb-PLGA-PLL-PEG-DNR group was significantly higher than that in DNR group（P<0.05）. TfR mAb-PLGA-PLL-PEG nanoparticle drug delivery system can target chemotherapy drugs to leukemia cells and enhance anticancer ability through apoptotic pathway.

Adsorptive Removal of Daunorubicin from Water by Graphene Oxide, Activated Carbon, and Multiwalled Carbon Nanotubes: Equilibrium and Kinetic Studies

AbstractThe adsorption of anticancer drug daunorubicin from aqueous solution by carbonaceous adsorbents was investigated. The adsorption performances of graphene oxide (GO), activated carbon (AC), and multiwalled carbon nanotubes (MWCNTs) were compared. The effects of solution pH, contact time, and initial daunorubicin concentration on the removal of daunorubicin were explored. Isotherm and kinetic models were applied to predict the efficiency of the adsorbents. The major functional groups of the adsorbents were identified by FTIR spectroscopy. In alkaline medium, adsorption increases with increasing pH value. With increasing initial concentration, adsorption capacity increases and removal efficiency decreases. Kinetic studies showed that the pseudo‐second‐order model describes adsorption onto GO well, whereas the intraparticle diffusion model gives the best fits for AC and MWCNTs. The adsorption isotherm fits the Langmuir model very well.

Tetrandrine overcomes drug resistance mediated by bone marrow microenvironment by regulating the expression of P-glycoprotein in acute leukemia

ABSTRACT Objectives: To study the effect of TET on the reversal of drug resistance in the bone marrow microenvironment, and to further promote the research on drug reversal.Methods: We established a co-culture system of bone marrow mesenchymal stem cells (BM-MSC) and K562 cell lines, and compared the cell inhibition rate of K562 cells between the co-culture group and K562 singleculture group by daunorubicin (DNR) single-drug intervention with CCK-8 and also compared K562 proliferation in the co-culture group and K562 single-culture group after combined intervention with DNR and TET, then used Western blot and RT-qPCR to verify the expression of P-gp of K562 cells at protein and mRNA levels, confirmed the concentration of DNR in K562 of different experimental groups by HPLC-MS.Results: According to the results of CCK-8, after co-culture with bone marrow mesenchymal stem cells (BM-MSCs), the inhibition rate of DNR on K562 decreased significantly. When TET (1μmol/L) combined with daunorubicin (DNR) treated on the co-culture group, the inhibition rate increased significantly. Then, the results of RT-qPCR and western blot showed a remarkable difference of the expression of P-glycoprotein (P-gp). After co-culture with BM-MSCs, the protein expression of P-gp showed a significant upward trend. After adding TET intervention, the expression of P-gp decreased both in mRNA and protein levels. Also, the DNR concentration in K562 also performed the correspondent trend.Conclusion: The bone marrow microenvironment can promote the MDR of acute leukemia. TET can reverse the MDR mediated by the bone marrow microenvironment by inhibiting the expression of P-glycoprotein.

CPX-351 Population Pharmacokinetics in Patients with Hematologic Malignancies

CPX-351, a dual-drug liposomal encapsulation of cytarabine and daunorubicin at a synergistic ratio, demonstrated significant survival benefit versus the conventional 7+3 regimen of cytarabine/daunorubicin in patients with secondary acute myeloid leukemia. A population pharmacokinetic (PK) analysis of total (ie, encapsulated + unencapsulated drug) plasma concentrations of cytarabine and daunorubicin following IV administration of CPX-351 assessed sources of variability in PK and determined if covariate-based dose adjustments may be warranted, particularly in patients with renal or hepatic impairment.The PK population consisted of 195 patients with advanced hematologic malignancies. The median age was 67 years (range: 24-81) and body weight was 79.8 kg (range: 38.9-156.5). There were 119 (61.0%) male and 76 (39.0%) female patients, mainly of white race (84.6%). A total of 83 (42.6%) patients had normal renal function (creatinine clearance [CrCL] ≥90 mL/min), 83 (42.6%) had mild renal impairment (CrCL 60-89 mL/min), 28 (14.4%) had moderate renal impairment (CrCL 30-59 mL/min), and 1 (0.5%) had severe renal impairment (CrCL 15-29 mL/min). The majority (n = 179 [91.8%]) of patients had bilirubin <1.2 mg/dL; the rest (n = 16 [8.2%]) had bilirubin between 1.2-3 mg/dL. No patient had end-stage renal disease or bilirubin >3 mg/dL, as expected since these patients are not usually considered fit for intensive chemotherapy.Nonlinear mixed-effect modeling was performed using NONMEM. Model evaluation and selection were assessed using a standard model discrimination process that included statistical criteria (eg, objective function value) and pertinent graphical representations of goodness-of-fit. Model validation/qualification was performed according to FDA and EMA guidelines for industry. Separate PK models were developed for cytarabine and daunorubicin, and intrinsic and extrinsic factors were evaluated as covariates. Intrinsic factors included body weight, body mass index, age, sex, race, white blood cell count, and markers of renal function (CrCL and serum creatinine) and hepatic function (bilirubin, aspartate and alanine aminotransferases, and alkaline phosphatase). The extrinsic factors were first dose amount, clinical study, and formulation.The base PK models for cytarabine and daunorubicin used 2-compartment structural models, with drug input into the central compartment, first-order distribution between the central and peripheral compartments, and first-order elimination from the central compartment. Two-compartment structural models with body surface area (BSA) as an allometric scalar provided minimum bias in estimates of systemic clearance (CL) and volume of distribution for the central compartment (Vc).Based on the final population PK model for cytarabine, a typical patient with BSA of 1.95 m2 receiving 100 units/m2 CPX-351 (100 mg/m2 cytarabine + 44 mg/m2 daunorubicin) is expected to have CL of 0.106 L/h with between-subject variability (BSV) of 70.5%, a Vc value of 5.12 L with BSV of 29.7%, and a peripheral volume of distribution (Vp) value of 0.214 L with BSV of 80.5%. Total volume of distribution (Vd = Vc + Vp) is predicted to be 5.33 L. The predicted half-lives associated with the distribution and elimination phases are 20.7 and 37.1 hours, respectively. The final population PK model for daunorubicin predicted a typical patient receiving the lyophilized formulation will have a CL value of 0.145 L/h with BSV of 49.4%, a Vc value of 4.34 L with BSV of 24.1%, and a Vp value of 0.523 L with BSV of 80.5%. Vd is predicted to be 4.86 L. The half-lives associated with the distribution and elimination phases are predicted to 11.1 and 26.2 hours, respectively.All covariates were eventually excluded from the population PK models, except dose and formulation, which had a small effect on PK and are not expected to result in detectable changes in clinical safety or efficacy. The PK of total cytarabine and daunorubicin were unaltered in patients with mild or moderate renal impairment (30 mL/min ≤ CrCL ≤ 89 mL/min). The potential effects of severe renal impairment could not be determined. Furthermore, the PK of total cytarabine and daunorubicin were unaltered in patients with serum bilirubin ≤3 mg/dL. In conclusion, the results of the population PK analysis indicated that dose adjustments of CPX-351 are not warranted for mild or moderate renal impairment or serum bilirubin up to 3 mg/dL. DisclosuresWang:Celator/Jazz: Employment, Equity Ownership. Banerjee:Celator/Jazz: Employment, Equity Ownership. Vasilinin:Celator/Jazz: Consultancy. Marier:Celator/Jazz: Consultancy. Gibbons:Celator/Jazz: Employment, Equity Ownership.

Acute Lymphoblastic Leukaemia (ALL) Cells and Commonly-Used Chemotherapy Drugs Can Induce Bone Marrow-Derived Mesenchymal Stromal Cells to Become Cancer Associated Fibroblasts (CAF), Leading to Enhanced ALL Proliferation and Treatment Resistance

To investigate the role of mesenchymal stromal cells (MSCs) in treatment resistance in acute lymphoblastic leukaemia (ALL) we systematically isolated and studied, ISCT-confirmed, bone-marrow-derived MSCs isolated from 151 adults (22-69 years) with ALL registered on the UKALL14 and ALL60+ trials both at diagnosis and during therapy.CD11b, CD68 and CD163-expressing tumour-associated macrophages were observed in 34/75 (45%) of MSC cultures from follow-up samples (termed M+MSC). By contrast, macrophages were absent (M-MSC) from all diagnostic (0/56) and healthy donor specimens (0/7) tested. ELISA on supernatant from expanded M+MSC showed significantly higher levels of pro-inflammatory cytokines IL8 (4961 pg/ml vs 1093 pg/ml, p = 0.01) and CXCL2 (106 pg/ml vs 36 pg/ml, p = 0.03) than that from expanded M-MSC. The B-ALL cell line Nalm6 proliferated more rapidly when co-cultured with primary M+MSCs than with primary M-MSC (1.93 fold, p = 0.03). Importantly, primary M+MSCs strongly expressed α-smooth muscle actin (αSMA) by immunostaining, consistent with a cancer associated fibroblast (CAF) phenotype.To establish the role of ALL cells and/or chemotherapy in the induction of CAFs, we used the MSC cell line HS27a to model CAF generation in-vitro . Co-culture of HS27a with B-ALL cell lines Nalm6, SD1 and 697 cells induced increased aSMA expression and cytoskeleton remodeling including increased actin stress fibers in the HS27a consistent with a CAF phenotype. These changes were not observed in HS27a in co-culture with healthy donor B-cells or the B-ALL cell line SEM. A targeted RQPCR gene panel showed up-regulation of CAF-defining genes (FAPA 2-4.4.6 fold, COX2 5.9 - 32.1 fold, IL6 11.2 - 495 fold, FN 2.2 - 3.0 fold, TGFβ1 2.0 - 4.6 fold, IL8 15.5 - 85.2 fold, CCL2 10.6 - 33.3 fold, VEGF 2.9 - 13.4 fold, FGF2 2.9 - 14.6 fold) and expected down-regulation of CAV1 (1.8 - 5.4 fold) when HS27a were cultured with SD1 and 697 cells compared to HS27a alone. HS27a exposed to healthy donor B-cells or SEM cells in contrast did not induce differential regulation of CAF defining genes. In a flow-based, cytokine bead assay, SD1 cells induced HS27a to secrete pro-inflammatory cytokines (IL8, IL6, CCL2, CXCL10 and CCL5) and growth factor bFGF at several fold higher levels than normal B-cells or SEM cells consistent with a CAF secretion profile. Similar morphological changes, gene expression profile and cytokine profile were observed when cells were co-cultured in a transwell system, suggesting a soluble mechanism of CAF induction. TGFβ1 and reactive oxygen species, known to induce CAFs in solid tumors, were both increased in all B-ALL cell lines able to induce CAFs compared to SEM cells, suggesting a probable mechanism behind CAF induction.Therapeutically-relevant concentrations of chemotherapy agents cytarabine and daunorubicin - but not vincristine and dexamethasone - induced gross morphological changes in HS27a cells with an increase in actin stress fibres and αSMA. RQPCR of cytarabine-treated HS27a showed upregulation of several genes consistent with a CAF phenotype and cytokine bead assay showed increased secretion of IL8 and CCL2 compared to untreated HS27a (973 pg/ml vs 430pg/ml, p < 0.0001 and 1089pg/ml vs 717.3pg/ml p = 0.02 respectively).To examine the functional significance of CAF on ALL cell proliferation and chemoresistance, we pre-treated HS27a with cytarabine to induce a CAF phenotype and then assessed the impact on SEM (selected for their lack of CAF induction) proliferation and chemosensitivity. Pretreatment with vincristine or dexamethasone and untreated HS27a comprised the controls. Cytarabine-generated, HS27a-derived CAF increased SEM cell proliferation compared to both the untreated and vincristine and dexamethasone pre-treated HS27a (1.2 fold p = 0.002, 1.3 fold p = 0.0004 and 1.2 fold p = 0.0002 respectively). Cytarabine-generated, HS27a-derived CAF also reduced the sensitivity of SEM cells to both dexamethasone and cytarabine compared to both the untreated and dexamethasone or vincristine pre-treated HS27a.We have demonstrated for the first time in both primary cells and cell-line models that bone-marrow derived MSCs can adopt a CAF phenotype in response to both ALL cells and ALL therapies. The functional impact is to enhance both ALL cell proliferation and chemotherapy resistance. Ongoing work will delineate the precise nature of the interaction between MSC and ALL cells. DisclosuresFielding:Baxalta: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees.

CPX-351 Population Pharmacokinetics in Pediatric and Adult Patients with Acute Myeloid Leukemia (AML)

CPX-351 (Vyxeos®; daunorubicin and cytarabine liposome for injection), a dual-drug liposomal encapsulation of daunorubicin and cytarabine at a 1:5 synergistic ratio, is approved by the US FDA and EMA for the treatment of adults with newly diagnosed therapy-related AML or AML with myelodysplasia-related changes. Recently, CPX-351 was used in pediatric patients (pts) with relapsed AML (JCO 2020, ASH 2019, ASH 2018). A population pharmacokinetic (PK) analysis of plasma concentrations of cytarabine and daunorubicin following IV administration of CPX-351 was performed to assess sources of variability in PK and to determine if age-based dose adjustments may be warranted, particularly in pediatric pts. The PK population consisted of 250 pts with advanced hematologic malignancies from 7 studies and included 46 (18%) pediatric pts (1-17 y) and 204 (82%) adults (≥18 y). The population included 148 (59%) males, mainly of white origin (82%). Nonlinear mixed-effect modeling was performed using NONMEM®. Model evaluation and selection were assessed using a standard model discrimination process that included statistical criteria (eg, objective function value) and pertinent graphical representations of goodness-of-fit. Separate PK models were developed for cytarabine and daunorubicin; intrinsic and extrinsic factors were evaluated as covariates. The intrinsic factors included body weight, body mass index, age, sex, race, white blood cell count, and markers of renal function (creatinine clearance, serum creatinine) and hepatic function (bilirubin, aspartate and alanine aminotransferases, alkaline phosphatase). The extrinsic factors included study type (adult vs pediatric/young adult) and formulation (frozen vs lyophilized). Based on previous population PK models developed for adults, the PK models for cytarabine and daunorubicin used 2-compartment structural models, with drug input into the central compartment, first-order distribution between the central and peripheral compartments, and first-order elimination from the central compartment. Two-compartment structural models with body surface area (BSA) as an allometric scalar provided minimum bias in estimates of systemic clearance (CL) and volume of distribution for the central compartment (Vc). Based on the final population PK models, the estimates of CL and Vc in adults were 0.101 L/h and 4.76 L, respectively, for cytarabine and 0.140 L/h and 4.04 L for daunorubicin. The population estimates of CL and Vc for cytarabine in children, adolescents, and young adults (&amp;lt;22 y) were 0.073 L/h and 3.91 L, respectively, for cytarabine and 0.093 L/h and 3.28 L for daunorubicin. The population PK models included an allometric component that accounted for differences in BSA. The exponent for the effect of BSA on CL was 0.948 and 0.876 for cytarabine and daunorubicin, respectively, suggesting a faster clearance in pts with higher BSA. These differences in CL are expected to be offset by BSA-based dosing of CPX-351. Bilirubin and formulation (frozen) remained statistically significant covariates on the CL of daunorubicin. All tested covariates were eventually excluded from the population PK models, except BSA, bilirubin, study type, and formulation, which have a small effect on PK and are not expected to result in detectable changes in clinical safety or efficacy. Age was evaluated both as a continuous and categorical variable (1-5, 6-11, and 12-17 y) and was not a significant covariate for cytarabine and daunorubicin CL and Vc. Study type was identified as a significant covariate for CL and Vc for cytarabine and daunorubicin, which was possibly due to a change in analytical site for pediatric studies. Mean AUC0-48 values of cytarabine in pts aged 1-5, 6-11, and 12-17 y (135 U/m2) were similar (2767, 2783, and 2806 μg·h/mL, respectively) and approximately 40% higher than that observed in pts ≥18 y (100 U/m2, 1928 μg·h/mL), proportional to the 35% higher dose in pediatric pts. Mean AUC0-48 values of daunorubicin in pts aged 1-5, 6-11, and 12-17 y (135 U/m2) were similar (967, 896, and 982 μg·h/mL, respectively) and approximately 40% higher than that observed in pts ≥18 y (100 U/m2, 615 μg·h/mL), proportional to the 35% higher dose in pediatric pts. The results of this population PK analysis indicated exposures to CPX-351 in pediatric pts were not affected by age and were similar to those in adults when administered at the same BSA-normalized dose. Disclosures Wang: Jazz Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Cooper:Celgene: Other: Spouse was an employee of Celgene (through August 2019). Absalon:Jazz Pharmaceuticals: Research Funding. OffLabel Disclosure: Yes, in this study, CPX-351 was also evaluated in pediatric AML

Pharmacokinetics, drug metabolism, and tissue distribution of CPX-351 in animals

CPX-351, a liposomal encapsulation of cytarabine and daunorubicin at a synergistic 5:1 molar ratio, is indicated for adults with newly diagnosed, therapy-related acute myeloid leukemia or acute myeloid leukemia with myelodysplasia-related changes. In preclinical species, this article demonstrated (1) similar release of cytarabine and daunorubicin by CPX-351 in plasma; (2) similar patterns of metabolism of cytarabine and daunorubicin following administration of CPX-351 versus non-liposomal cytarabine/daunorubicin combination; (3) prolonged tissue exposure to CPX-351; (4) dramatically different tissue distribution of cytarabine and daunorubicin following administration of CPX-351 versus non-liposomal combination (tissue:plasma ratios generally <1 versus >1, respectively); and (5) dramatically lower unbound plasma and tissue concentrations of cytarabine and daunorubicin following administration of CPX-351 versus non-liposomal combination. Together, these results provide insight into the safety profile of CPX-351, as well as mechanisms that drive the improved efficacy observed for CPX-351 versus the conventional 7 + 3 cytarabine/daunorubicin regimen in clinical studies.

The epigallocatechin gallate derivative Y6 reduces the cardiotoxicity and enhances the efficacy of daunorubicin against human hepatocellular carcinoma by inhibiting carbonyl reductase 1 expression

Ethnopharmacological relevanceGreen tea is the most ancient and popular beverage worldwide and its main constituent epigallocatechin-3-gallate (EGCG) has a potential role in the management of cancer through the modulation of cell signaling pathways. However, EGCG is frangible to oxidation and exhibits low lipid solubility and bioavailability, and we synthesized a derivative of EGCG in an attempt to overcome these limitations. Aim of the studyThe anthracycline antibiotic daunorubicin (DNR) is a potent anticancer agent. However, its severe cardiotoxic limits its clinical efficacy. Human carbonyl reductase 1 (CBR1) is one of the most effective human reductases for producing hydroxyl metabolites and thus may be involved in increasing the cardiotoxicity and decreasing the antineoplastic effect of anthracycline antibiotics. Accordingly, in this study, we investigated the co-therapeutic effect of Y6, a novel and potent adjuvant obtained by optimization of the structure of EGCG. Material and methodsThe cellular concentrations of DNR and its metabolite DNRol were measured by HPLC to determine the effects of EGCG and Y6 on the inhibition of DNRol formation. The cytotoxic effects of EGCG and Y6 were tested by MTT assay in order to identify non-toxic concentrations of them. To understand their antitumor and cardioprotective mechanisms, hypoxia-inducible factor-1α (HIF-1α) and CBR1 protein expression was measured via Western blotting and immunohistochemical staining while gene expression was analyzed using RT-PCR. Moreover, PI3K/AKT and MEK/ERK signaling pathways were analyzed via Western blotting. HepG2 xenograft model was used to detect the effects of EGCG and Y6 on the antitumor activity and cardiotoxicity of DNR in vivo. Finally, to obtain further insight into the interactions of Y6 and EGCG with HIF-1α and CBR1, we performed a molecular modeling. ResultsY6(10 μg/ml or 55 mg/kg) decreased the expression of HIF-1α and CBR1 at both the mRNA and protein levels during combined drug therapy in vitro as well as in vivo, thereby inhibiting formation of the metabolite DNRol from DNR, with the mechanisms being related to PI3K/AKT and MEK/ERK signaling inhibition. In a human carcinoma xenograft model established with subcutaneous HepG2 cells, Y6(55 mg/kg) enhanced the antitumor effect and reduced the cardiotoxicity of DNR more effectively than EGCG(40 mg/kg). ConclusionsY6 has the ability to inhibit CBR1 expression through the coordinate inhibition of PI3K/AKT and MEK/ERK signaling, then synergistically enhances the antitumor effect and reduces the cardiotoxicity of DNR.

Can image analysis provide evidence that lysosomal sequestration mediates daunorubicin resistance?

Altered intracellular distribution of weak base anticancer drugs owing to lysosomal sequestration is one purported mechanism contributing to chemotherapy resistance. This has often been demonstrated with the example of daunorubicin (DNR), chemotherapy with its characteristic red fluorescence used to trace it in cellular compartments.Here we addressed the question whether image analysis of DNR fluorescence can reflect its real intracellular distribution. We observed that the relationship between the intensity of the DNR fluorescence and its concentration in water solutions with or without proteins is far from linear. In contrast, nucleic acids, RNA and DNA in particular, dramatically diminish the DNR fluorescence, however, the intensity was proportional to the amount. Therefore, image analysis reflects the composition of different cell compartments (i.e., the presence of proteins and nucleic acids) rather than the actual concentration of DNR in these compartments. In line with these results, we observed highly fluorescent lysosomes and low fluorescent nucleus in sensitive cancer cells treated with low DNR concentrations, a fluorescence pattern thought to be found only in resistant cancer cells. Importantly, LC/MS/MS analysis of extracts from sensitive cells treated with DNR or DNR in combination with an inhibitor of vacuolar ATPase, concanamycin A, indicated that lysosomal accumulation of DNR increased with increasing extracellular concentration. However, even the highest lysosomal accumulation of DNR failed to reduce its extralysosomal concentration and thus change the cell sensitivity to the drug.In conclusion, our results strongly suggest that DNR fluorescence within cells does not indicate the real drug distribution. Further they suggested that lysosomal sequestration of DNR can hardly contribute to its resistance in cancer cells in vitro.

Effect of daunorubicin on acute promyelocytic leukemia cells using nuclear magnetic resonance spectroscopy-based metabolomics

The aim of this study was to determine several key metabolites as potential biomarkers of daunorubicin (DNR) treatment of acute promyelocytic leukemia (APL) using APL blasts and NB4 cells. Samples which were obtained from 16 newly diagnosed APL patients and human APL NB4 cell lines were exposed to increasing concentrations of DNR (0 μM, 0.1 μM, 0.5 μM and 1.0 μM). Electron microscopy and Nuclear Magnetic Resonance (NMR) spectroscopy confirmed that there were clear differences between controls and DNR-treated groups, with the resultant models having excellent predictive and discriminative abilities. Four metabolites meeting the biomarker requirements were identified. KEGG analyses revealed that these biomarkers were associated with the metabolism of fat, choline, and glucose. These findings offered vital information about the effects of chemotherapies on the whole body biochemistry which might be important for monitoring apoptosis and injury to cells in order to reduce chemotherapies-induced side effects.

Carbon quantum dot modified electrodes developed for electrochemical monitoring of Daunorubicin-DNA interaction

The carbon quantum dot (cQD) modified disposable pencil graphite electrodes were developed for the first time in this study for electrochemical monitoring of drug-DNA interaction. The biomolecular interaction between calf thymus double stranded DNA (ctDNA) and an anthracycline antineoplastic drug, Daunorubicin (DNR) was investigated by differential pulse voltammetry (DPV) technique. For this purpose, experimental conditions, such as, the concentration of ctDNA, concentration of DNR and interaction time were optimized. Under optimum conditions, the detection limits of DNR and ctDNA were found to be 0.02 μg/mL and 0.89 μg/mL, respectively. The effect of interaction time between DNR and ctDNA was explored upon to the changes at both DNR and guanine oxidation signals. Drug-DNA interaction process was also examined by electrochemical impedance spectroscopy (EIS) technique.

Daunorubicin-Loaded CdTe QDs Conjugated with Anti-CD123 mAbs: A Novel Delivery System for Myelodysplastic Syndromes Treatment.

IntroductionThe myelodysplastic syndromes (MDS) are a very heterogeneous group of myeloid disorders characterized by peripheral blood cytopenias and increase risk of transformation to acute myeloid leukemia (AML). Daunorubicin (DNR) is an indispensable drug for the treatment of MDS and AML. However, its side effects including cardiac toxicity and bone marrow suppression severely limit clinical application. Many researches reported high expression of CD123 antigen on high-risk MDS cells, so we constructed a novel drug delivery system comprising daunorubicin-loaded CdTe QDs conjugated with anti-CD123 mAbs (DNR-CdTe-CD123) to develop targeted combination chemotherapy for MDS.MethodsCdTe conjugated antiCD123 through amide bond, co-loaded with DNR with electrostatic bonding. Then, we determined characterization and release rate of DNR-CdTe-CD123. The therapeutic effect and side effect of drug delivery system were evaluated through in vitro and in vivo experiments.ResultsCdTe showed appropriate diameter and good dispersibility and DNR was loaded into CdTes with high encapsulation efficiency and drug loading. The maximum drug loading and encapsulation efficiency were 42.08 ± 0.64% and 74.52 ± 1.81%, respectively, at DNR concentration of 0.2mg/mL and anti-CD123 mAbs volume of 5ul (100ug/mL). Flow cytometry (FCM) showed that CD123 antigen was highly expressed on MUTZ-1 cells, and its expression rate was 72.89 ± 10.67%. In vitro experiments showed that the inhibition rate and apoptosis rate of MUTZ-1 cells treated with DNR-CdTe-CD123 were higher than those in the other groups (P<0.05). Compared with the other groups, the level of apoptosis-related protein (P53, cleaved caspase-9, Bax and cleaved caspase-3) were upregulated in DNR-CdTe-CD123 group (P<0.05). In vivo experiments, DNR-CdTe-CD123 can effectively inhibit the tumor growth of MDS-bearing nude mice and reduce the side effects of DNR on myocardial cells.ConclusionThe system of DNR-CdTe-CD123 enhances the therapeutic effects and reduce the side effects of DNR, thus providing a novel platform for MDS treatment.

Daunorubicin-containing CLL1-targeting nanomicelles have anti-leukemia stem cell activity in acute myeloid leukemia

Patients with acute myeloid leukemia have a very poor prognosis related to a high rate of relapse and drug-related toxicity. The ability of leukemia stem cells (LSCs) to survive chemotherapy is primarily responsible for relapse, and eliminating LSCs is ultimately essential for cure. We developed novel disulfide-crosslinked CLL1-targeting micelles (DC-CTM), which can deliver high concentrations of daunorubicin (DNR) into both bulk leukemia cells and LSCs. Compared to free DNR, DC-CTM-DNR had a longer half-life, increased DNR area under the curve concentration by 11-fold, and exhibited a superior toxicity profile. In patient-derived AML xenograft models, DC-CTM-DNR treatment led to significant decreases in AML engraftment and impairment of secondary transplantation compared to control groups. Collectively, we demonstrate superior anti-LSC/AML efficacy, and preferable pharmacokinetic and toxicity profiles of DC-CTM-DNR compared to free DNR. DC-CTM-DNR has the potential to significantly improve treatment outcomes and reduce therapy-related morbidity and mortality for patients with AML.

Applications of daunorubicin-loaded PLGA-PLL-PEG-Tf nanoparticles in hematologic malignancies: an in vitro and in vivo evaluation.

BackgroundWith the development of drug delivery, novel tools and technological approaches have captured the attention of researchers in recent years. Several target drug delivery systems (DDSs) including nanoparticles (NPs) have been developed as an important strategy to deliver classical medicine.ObjectiveThe objective of this study was to evaluate the application of novel daunorubicin (DNR)-loaded poly(lactic-co-glycolic acid)-poly-l-lysine-polyethylene glycol-transferrin (Tf) nanoparticles (DNR-loaded NPs) in hematologic malignancies in vitro and in vivo.Materials and methodsDNR-loaded NPs were prepared by the modified double-emulsion solvent evaporation/diffusion method, and its microscopic form was observed under scanning electron microscope. Intracellular distribution of DNR was directly detected by fluorescence microscopy. After establishment of a tumor xenograft model by injecting K562 cells into the left leg of nude mice, the therapeutic effect of the DNR-loaded NPs on the growth of tumors was measured by calculating the tumor size, and the relative expression of Caspase-3 protein was detected by immunohistochemical staining. Furthermore, intracellular concentration of DNR and the extent of cell apoptosis in primary leukemia cells were quantified by flow cytometry.ResultsDNR-loaded NPs had a spherical shape of about 180 nm in diameter. DNR-loaded NP group showed a significant enhancement of cellular uptake in K562 cells compared with DNR group. Tumor inhibition rate was higher in DNR-loaded NP group in comparison with DNR group, and the relative expression of Caspase-3 protein was upregulated in DNR-loaded NP group compared with DNR group. Furthermore, DNR-loaded NPs obviously increased intracellular concentration of DNR in primary leukemia cells compared with DNR group, but there was no significant difference in primary cell apoptosis between the two groups. These findings suggest that the novel NP DDS can enhance the performance of conventional antitumor drugs and may be suitable for further application in the treatment of hematologic malignancies.

Personalized and Optimized Low-Dose and Intensive Chemotherapy Treatments for Patients with Acute Myeloid Leukemia (AML)

Patients with AML have heterogeneous features, including those specific to the patient as well as those specific to the disease, such as leukemic burden and dynamic sub-clonal populations. Outside of clinical trials, few of these components are used to determine treatment. In order to move towards precision medicine, we have developed πiChemo, a computational application based on a dynamic mathematical modelling framework, using patient-, leukemia- and treatment-specific data to predict outcomes and optimize chemotherapy regimens for patients with AML.The model consists of a pharmacokinetic and pharmacodynamic (PK/PD) module that calculates the concentration and effect of Cytarabine Arabinoside (Ara-C) and Daunorubicin (DNR) in bone marrow (BM); and a population balance models (PBMs) module that describes normal populations (stem cells, progenitors, precursors) and abnormal populations (leukemic sensitive blasts (LSB) and leukemic resistant blasts (LRB)) in BM. The PBMs module also determines mature cell numbers in three lineages found in BM and peripheral blood (PB): (1) red blood cells (RBC), (2) white blood cells (WBC) and (3) lymphocytes (L). Model structure was analysed by global sensitivity analysis, which identified the most significant parameters on outcome predictions, re-estimated for each patient. The final integrated PK/PD & PBMs model has 1,295 differential equations, 8,044 algebraic equations, 9,335 variables, 25 fixed parameters and 4 degrees of freedom or variables to be optimized (Ara-C dose, Ara-C injection duration, DNR dose and DNR injection duration).Model validation, predictions and optimizations were performed using anonymised retrospective data from 28 patients with AML. The model required: (i) patient features: height, weight, age and gender, (ii) patient status: initial BM differential and PB cell counts, (iii) leukemia data: cellularity, presence of dysplasia and initial blast percentage and, (iv) treatment data: type (low-dose (LD) or intensive (DA)), dose, administration route (SC vs IV), administration mode (bolus injection vs infusion), time between injections and between cycles.The model predicted the absolute numbers of stem cells, progenitors, precursors, WBC, RBC, L, LSB and LRB in BM, and WBC, RBC, L and neutrophil count in PB during treatment for all patients. Model simulations predicted outcomes for 18 patients who achieved complete remission (7 LD & 11 DA), 4 patients who entered partial remission (2 LD & 2 DA) and 6 patients who relapsed (2 LD & 4 DA). The most remarkable results are those of prediction for BM blast percentage after each chemotherapy cycle and the PB neutrophil count for all patients. The notable fit between model predictions and daily patient data demonstrate model robustness and accuracy in the capacity to track patient-specific restaging BM and daily PB count evolution before, during and after treatment.The same patient datasets were used to apply an optimization algorithm that could maximize normal cell number and reduce leukemia burden, to personalize chemotherapy dose and administration for best outcomes. The results show that doses and administration methods vary between patients and between chemotherapy cycles for the same patient, depending on the evolution of normal and abnormal populations in BM. Low-dose continuous Ara-C infusions were more effective than rapid bolus injections, due to reduced chemotherapy effects on normal cells and subsequent quicker recovery in the normal BM compartments. RBC progenitors and precursors recovered faster than WBC and L lineages, and the recovery of normal BM cells was faster than that of normal mature cells in PB.The πiChemo tool requires only patient- and leukemia-specific initial conditions at diagnosis, easily obtained in standard clinical practice, for outcome predictions and treatment optimizations. Real-time model-fit testing and comparison of model results against daily PB cell counts would enable the re-estimation of significant parameters, increasing model accuracy and treatment effectiveness whilst therapy is ongoing. The πiChemo precision therapy tool has the potential to personalize optimal standard and novel treatments for AML in real-time. DisclosuresNo relevant conflicts of interest to declare.

Porous silicon based intravitreal platform for dual-drug loading and controlled release towards synergistic therapy

The number of blind and low vision persons in the US is projected to increase to 5.68 million by 2020. The eye diseases causing loss of vision are life-long, chronic, and often need protracted presence of therapeutics at the disease site to keep the disease in remission. In addition, multiple pathologies participate in the disease process and a single therapy seems insufficient to bring the disease under control and prevent vision loss. This study demonstrates the use of porous silicon (pSi) particles sequentially loaded with daunorubicin (DNR) and dexamethasone (DEX) to create a synergistic intravitreally injectable dual-drug delivery system. DEX targets chronic inflammation while DNR inhibits excessive cell proliferation as well as suppresses hypoxia-inducible factor 1 to reduce scarring. This pSi-based delivery system releases therapeutic concentrations of DNR for 100 days and DEX for over 165 days after a single dose. This intravitreal dual-drug delivery system is also well tolerated after injection into the rabbit eye model, attested by ocular biomicroscopy, ocular tonometry, electroretinography, and histology. This novel dual-drug delivery system opens an attractive modality for combination therapy to manage refractory chorioretinal diseases and further preclinical studies are warranted to evaluate its efficacy.

Adipocytes Sequester and Metabolize the Chemotherapeutic Daunorubicin

Obesity is associated with poorer outcome for many cancers. Previously, we observed that adipocytes protect acute lymphoblastic leukemia (ALL) cells from the anthracycline, daunorubicin. In this study, it is determined whether adipocytes clear daunorubicin from the tumor microenvironment (TME). Intracellular daunorubicin concentrations were evaluated using fluorescence. Daunorubicin and its largely inactive metabolite, daunorubicinol, were analytically measured in media, cells, and tissues using liquid chromatography/mass spectrometry (LC/MS). Expression of daunorubicin-metabolizing enzymes, aldo-keto reductases (AKR1A1, AKR1B1, AKR1C1, AKR1C2, AKR1C3, and AKR7A2) and carbonyl reductases (CBR1, CBR3), in human adipose tissue, were queried using public databases and directly measured by quantitative PCR (qPCR) and immunoblot. Adipose tissue AKR activity was measured by colorimetric assay. Adipocytes absorbed and efficiently metabolized daunorubicin to daunorubicinol, reducing its antileukemia effect in the local microenvironment. Murine studies confirmed adipose tissue conversion of daunorubicin to daunorubicinol in vivo Adipocytes expressed high levels of AKR and CBR isoenzymes that deactivate anthracyclines. Indeed, adipocyte protein levels of AKR1C1, AKR1C2, and AKR1C3 are higher than all other human noncancerous cell types. To our knowledge, this is the first demonstration that adipocytes metabolize and inactivate a therapeutic drug. Adipocyte-mediated daunorubicin metabolism reduces active drug concentration in the TME. These results could be clinically important for adipocyte-rich cancer microenvironments such as omentum, breast, and marrow. As AKR and CBR enzymes metabolize several drugs, and can be expressed at higher levels in obese individuals, this proof-of-principle finding has important implications across many diseases.Implications: Adipocyte absorption and metabolism of chemotherapies can reduce cytotoxicity in cancer microenvironments, potentially contributing to poorer survival outcomes. Mol Cancer Res; 15(12); 1704-13. ©2017 AACR.

Ag-4-ATP-MWCNT electrode modified with dsDNA as label-free electrochemical sensor for the detection of daunorubicin anticancer drug

This paper describes the synthesis of Ag-4-ATP-MWCNT nanocomposite and its use as a modifier of working electrode. The surface of the electrochemical Ag-4-ATP-MWCNT electrode was modified with a double-stranded DNA (dsDNA) to detect daunorubicin-DNA interactions. Differential pulse voltammetry was applied to develop an electroanalytical procedure for the determination of daunorubicin and evaluate its interaction with dsDNA immobilized on the electrode surface. After the optimization of operational parameters, the linear dependence of the peak current on the daunorubicin concentration was observed in the range of 0.10×10−8 to 1.00×10−5molL−1, with the detection and quantification limits of 3.00×10−10 and 1.00×10−9molL−1, respectively. The proposed biosensor was successfully applied to validate its capability for the determination of daunorubicin in human serum and urine samples.