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A liposomal formulation of the synthetic curcumin analog EF24 (Lipo-EF24) inhibits pancreatic cancer progression: towards future combination therapies.

Bisht S, Schlesinger M, Rupp A, Schubert R, Nolting J, Wenzel J, Holdenrieder S, Brossart P, Bendas G, Feldmann G - J Nanobiotechnology (2016)

Bottom Line: Lipo-EF24 potently suppressed NF-kappaB nuclear translocation by inhibiting phosphorylation and subsequent degradation of its inhibitor I-kappa-B-alpha.In vivo, synergistic tumor growth inhibition was observed in MIAPaCa xenografts when Lipo-EF24 was given in combination with the standard-of-care cytotoxic agent gemcitabine.In line with in vitro observations, western blot analysis revealed decreased phosphorylation of I-kappa-B-alpha in excised Lipo-EF24-treated xenograft tumor tissues.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine 3, Center of Integrated Oncology (CIO) Cologne-Bonn, University Hospital of Bonn, Sigmund-Freud-Str. 25, 53127, Bonn, Germany.

ABSTRACT

Background: Pancreatic cancer is one of the most lethal of human malignancies known to date and shows relative insensitivity towards most of the clinically available therapy regimens. 3,5-bis(2-fluorobenzylidene)-4-piperidone (EF24), a novel synthetic curcumin analog, has shown promising in vitro therapeutic efficacy in various human cancer cells, but insufficient water solubility and systemic bioavailability limit its clinical application. Here, we describe nano-encapsulation of EF24 into pegylated liposomes (Lipo-EF24) and evaluation of these particles in preclinical in vitro and in vivo model systems of pancreatic cancer.

Results: Transmission electron microscopy and size distribution studies by dynamic light scattering confirmed intact spherical morphology of the formed liposomes with an average diameter of less than 150 nm. In vitro, treatment with Lipo-EF24 induced growth inhibition and apoptosis in MIAPaCa and Pa03C pancreatic cancer cells as assessed by using cell viability and proliferation assays, replating and soft agar clonogenicity assays as well as western blot analyses. Lipo-EF24 potently suppressed NF-kappaB nuclear translocation by inhibiting phosphorylation and subsequent degradation of its inhibitor I-kappa-B-alpha. In vivo, synergistic tumor growth inhibition was observed in MIAPaCa xenografts when Lipo-EF24 was given in combination with the standard-of-care cytotoxic agent gemcitabine. In line with in vitro observations, western blot analysis revealed decreased phosphorylation of I-kappa-B-alpha in excised Lipo-EF24-treated xenograft tumor tissues.

Conclusion: Due to its promising therapeutic efficacy and favorable toxicity profile Lipo-EF24 might be a promising starting point for development of future combinatorial therapeutic regimens against pancreatic cancer.

No MeSH data available.


Related in: MedlinePlus

In vivo pharmacokinetics of EF24 loaded liposomes. a Graphical representation of the results from LC–MS/MS measurements. Left linear scale; right semilogarithmic scale. Plasma concentrations of EF24 were determined at 0.25, 1, 2, 3, 4, 6, 8 and 24 h after intravenous administration of liposomal EF24 formulation, given as single dose equivalent to 10 mg/kg EF24. For each time point data from at least two to four mice were analysed. b Graphical representation of the results of free (not encapsulated) EF24 solubilized in DMSO from LC–MS/MS measurements. Left linear scale; right semilogarithmic scale
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Fig7: In vivo pharmacokinetics of EF24 loaded liposomes. a Graphical representation of the results from LC–MS/MS measurements. Left linear scale; right semilogarithmic scale. Plasma concentrations of EF24 were determined at 0.25, 1, 2, 3, 4, 6, 8 and 24 h after intravenous administration of liposomal EF24 formulation, given as single dose equivalent to 10 mg/kg EF24. For each time point data from at least two to four mice were analysed. b Graphical representation of the results of free (not encapsulated) EF24 solubilized in DMSO from LC–MS/MS measurements. Left linear scale; right semilogarithmic scale

Mentions: The mice were injected intravenously with a single dose of encapsulated EF24 (10 mg/kg) at t = 0 h. Peripheral blood samples drawn at different time intervals (0.25, 1, 2, 3, 4, 6, 8 and 24 h, respectively) were quantified by means of LC–MS/MS and the results obtained were averaged afterwards. The data thus acquired were plotted on a linear and semi-logarithmic scale (Fig. 7). As evident from these graphs, a rapid initial decrease in mean EF24 plasma concentrations within the first hour was followed by a subsequent phase of less pronounced decline between t = 1 h and t = 8 h. At 24 h, mean plasma levels of EF24 were found to be below the detection limit. Thus, a distribution phase within the first hours and an elimination phase (after t = 1 h) could be differentiated. Both phases were fitted separately on a semi-logarithmic scale, which yielded rate constants of kdist = 4.25 h−1 and kelim = 0.31 h−1, respectively. Corresponding half-life values were t1/2,dist = 0.16 h and t1/2,elim = 2.23 h. Due to sharp initial concentration decrease, the area under the concentration–time curve (AUC) was determined by both linear and logarithmic trapezoidal method, which yielded corresponding values of AUClin = 3074 ng h L−1 and AUClog = 2456 ng h L−1, respectively. Upon extrapolation from t = 0.25 h to t = 0 h, values amount to AUClin,0 = 4197 ng h L−1 and AUClog,0 = 3573 ng h L−1, respectively. Additionally, extrapolating the concentration c to t = 0 h results in cmax = 5023 ng L−1 (Fig. 7a). For comparison, the pharmacokinetic profile of pure EF24 solubilized in DMSO was also determined by LC–MS/MS in the time regime between 0.25 and 8 h after intravenous injection of a single dose of EF24 (10 mg kg−1). Without the liposomic encapsulation EF24 shows no sign of a distribution phase, but is constantly eliminated with a rate constant of 0.4683 h−1 and a half-life of t1/2 = 2.14 h. The measured peak concentration at t = 0.25 h is roughly 75 % of the peak concentration of encapsulated EF24. Extrapolating the first three concentration time points to t = 0 yields an initial concentration of roughly 5500 ng L−1, which is slightly higher than in the case of EF24 encapsulated in liposomes. Accordingly, the AUC also amounts to a higher value of 5258 ng L−1 h−1 (Fig. 7b).Fig. 7


A liposomal formulation of the synthetic curcumin analog EF24 (Lipo-EF24) inhibits pancreatic cancer progression: towards future combination therapies.

Bisht S, Schlesinger M, Rupp A, Schubert R, Nolting J, Wenzel J, Holdenrieder S, Brossart P, Bendas G, Feldmann G - J Nanobiotechnology (2016)

In vivo pharmacokinetics of EF24 loaded liposomes. a Graphical representation of the results from LC–MS/MS measurements. Left linear scale; right semilogarithmic scale. Plasma concentrations of EF24 were determined at 0.25, 1, 2, 3, 4, 6, 8 and 24 h after intravenous administration of liposomal EF24 formulation, given as single dose equivalent to 10 mg/kg EF24. For each time point data from at least two to four mice were analysed. b Graphical representation of the results of free (not encapsulated) EF24 solubilized in DMSO from LC–MS/MS measurements. Left linear scale; right semilogarithmic scale
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Related In: Results  -  Collection

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Fig7: In vivo pharmacokinetics of EF24 loaded liposomes. a Graphical representation of the results from LC–MS/MS measurements. Left linear scale; right semilogarithmic scale. Plasma concentrations of EF24 were determined at 0.25, 1, 2, 3, 4, 6, 8 and 24 h after intravenous administration of liposomal EF24 formulation, given as single dose equivalent to 10 mg/kg EF24. For each time point data from at least two to four mice were analysed. b Graphical representation of the results of free (not encapsulated) EF24 solubilized in DMSO from LC–MS/MS measurements. Left linear scale; right semilogarithmic scale
Mentions: The mice were injected intravenously with a single dose of encapsulated EF24 (10 mg/kg) at t = 0 h. Peripheral blood samples drawn at different time intervals (0.25, 1, 2, 3, 4, 6, 8 and 24 h, respectively) were quantified by means of LC–MS/MS and the results obtained were averaged afterwards. The data thus acquired were plotted on a linear and semi-logarithmic scale (Fig. 7). As evident from these graphs, a rapid initial decrease in mean EF24 plasma concentrations within the first hour was followed by a subsequent phase of less pronounced decline between t = 1 h and t = 8 h. At 24 h, mean plasma levels of EF24 were found to be below the detection limit. Thus, a distribution phase within the first hours and an elimination phase (after t = 1 h) could be differentiated. Both phases were fitted separately on a semi-logarithmic scale, which yielded rate constants of kdist = 4.25 h−1 and kelim = 0.31 h−1, respectively. Corresponding half-life values were t1/2,dist = 0.16 h and t1/2,elim = 2.23 h. Due to sharp initial concentration decrease, the area under the concentration–time curve (AUC) was determined by both linear and logarithmic trapezoidal method, which yielded corresponding values of AUClin = 3074 ng h L−1 and AUClog = 2456 ng h L−1, respectively. Upon extrapolation from t = 0.25 h to t = 0 h, values amount to AUClin,0 = 4197 ng h L−1 and AUClog,0 = 3573 ng h L−1, respectively. Additionally, extrapolating the concentration c to t = 0 h results in cmax = 5023 ng L−1 (Fig. 7a). For comparison, the pharmacokinetic profile of pure EF24 solubilized in DMSO was also determined by LC–MS/MS in the time regime between 0.25 and 8 h after intravenous injection of a single dose of EF24 (10 mg kg−1). Without the liposomic encapsulation EF24 shows no sign of a distribution phase, but is constantly eliminated with a rate constant of 0.4683 h−1 and a half-life of t1/2 = 2.14 h. The measured peak concentration at t = 0.25 h is roughly 75 % of the peak concentration of encapsulated EF24. Extrapolating the first three concentration time points to t = 0 yields an initial concentration of roughly 5500 ng L−1, which is slightly higher than in the case of EF24 encapsulated in liposomes. Accordingly, the AUC also amounts to a higher value of 5258 ng L−1 h−1 (Fig. 7b).Fig. 7

Bottom Line: Lipo-EF24 potently suppressed NF-kappaB nuclear translocation by inhibiting phosphorylation and subsequent degradation of its inhibitor I-kappa-B-alpha.In vivo, synergistic tumor growth inhibition was observed in MIAPaCa xenografts when Lipo-EF24 was given in combination with the standard-of-care cytotoxic agent gemcitabine.In line with in vitro observations, western blot analysis revealed decreased phosphorylation of I-kappa-B-alpha in excised Lipo-EF24-treated xenograft tumor tissues.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine 3, Center of Integrated Oncology (CIO) Cologne-Bonn, University Hospital of Bonn, Sigmund-Freud-Str. 25, 53127, Bonn, Germany.

ABSTRACT

Background: Pancreatic cancer is one of the most lethal of human malignancies known to date and shows relative insensitivity towards most of the clinically available therapy regimens. 3,5-bis(2-fluorobenzylidene)-4-piperidone (EF24), a novel synthetic curcumin analog, has shown promising in vitro therapeutic efficacy in various human cancer cells, but insufficient water solubility and systemic bioavailability limit its clinical application. Here, we describe nano-encapsulation of EF24 into pegylated liposomes (Lipo-EF24) and evaluation of these particles in preclinical in vitro and in vivo model systems of pancreatic cancer.

Results: Transmission electron microscopy and size distribution studies by dynamic light scattering confirmed intact spherical morphology of the formed liposomes with an average diameter of less than 150 nm. In vitro, treatment with Lipo-EF24 induced growth inhibition and apoptosis in MIAPaCa and Pa03C pancreatic cancer cells as assessed by using cell viability and proliferation assays, replating and soft agar clonogenicity assays as well as western blot analyses. Lipo-EF24 potently suppressed NF-kappaB nuclear translocation by inhibiting phosphorylation and subsequent degradation of its inhibitor I-kappa-B-alpha. In vivo, synergistic tumor growth inhibition was observed in MIAPaCa xenografts when Lipo-EF24 was given in combination with the standard-of-care cytotoxic agent gemcitabine. In line with in vitro observations, western blot analysis revealed decreased phosphorylation of I-kappa-B-alpha in excised Lipo-EF24-treated xenograft tumor tissues.

Conclusion: Due to its promising therapeutic efficacy and favorable toxicity profile Lipo-EF24 might be a promising starting point for development of future combinatorial therapeutic regimens against pancreatic cancer.

No MeSH data available.


Related in: MedlinePlus