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Pharmaceutical Research

, Volume 31, Issue 9, pp 2276–2286 | Cite as

Gemcitabine Treatment of Rat Soft Tissue Sarcoma with Phosphatidyldiglycerol-Based Thermosensitive Liposomes

  • Simone Limmer
  • Jasmin Hahn
  • Rebecca Schmidt
  • Kirsten Wachholz
  • Anja Zengerle
  • Katharina Lechner
  • Hansjörg Eibl
  • Rolf D. Issels
  • Martin Hossann
  • Lars H. Lindner
Research Paper

Abstract

Purpose

The pyrimidine analogue gemcitabine (dFdC) is frequently used in the treatment of patients with solid tumors. However, after i.v. application dFdC is rapidly inactivated by metabolization. Here, the potential of thermosensitive liposomes based on 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2-TSL) were investigated as carrier and targeting system for delivery of dFdC in combination with local hyperthermia (HT).

Methods

DPPG2-TSL were prepared by the lipid film hydration and extrusion method and characterized by dynamic light scattering, thin layer chromatography, phosphate assay and HPLC. In vivo experiments were performed in Brown Norway rats with a syngeneic soft tissue sarcoma. Local HT treatment was performed by light exposure.

Results

DPPG2-TSL were stable at 37°C in serum and showed a temperature dependent dFdC release >40°C. Plasma half-life of dFdC was strongly increased from 0.07 h (non-liposomal) to 0.53 h (liposomal, vesicle size 105 nm) or 2.59 h (liposomal, 129 nm). Therapy of BN175 tumors with dFdC encapsulated in DPPG2-TSL + HT showed significant improvement in tumor growth delay compared to non-liposomal dFdC without HT (p < 0.05), non-liposomal dFdC with HT (p < 0.01), and liposomal dFdC without HT (p < 0.05), respectively.

Conclusions

Gemcitabine encapsulated in DPPG2-TSL in combination with local HT is a promising tool for the treatment of solid tumors. Therefore, these encouraging results ask for further investigation and evaluation.

Key words

drug delivery gemcitabine hyperthermia phosphatidyloligoglycerol thermosensitive liposomes 

Abbreviations

dFdC

gemcitabine

dFdCTP

gemcitabine triphosphate

DLS

dynamic light scattering

DPPC

1,2-dipalmitoyl-sn-glycero-3-phosphocholine

DPPG2

1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol

DPPG2-TSL

liposomes composed of DPPC/DSPC/DPPG2 50/20/30 (mol/mol)

DSPC

1,2-distearoyl-sn-glycero-3-phosphocholine

FCS

fetal calf serum

HPLC

high performance liquid chromatography

HT

local hyperthermia

i.v.

intravenous

IS

internal standard

Lyso-PC

lyso-phosphatidylcholine

Lyso-PG2

lyso-phosphatidyldiglycerol

P-lyso-PC

1-palmitoyl-sn-glycero-3-phosphocholine

TLC

thin layer chromatography

Tm

solid gel to liquid disordered phase transition temperature

TSL

thermosensitive liposomes

Notes

Acknowledgments And Disclosures

The authors gratefully acknowledge the help of E. Wagner (Department of Pharmaceutical Biology-Biotechnology, Ludwig-Maximilians University, Munich, Germany) for providing facilities.

The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 603028 (iPaCT project).

Chemical compounds studied in this study: DSPC (Pubmed CID 94190), DPPC (Pubmed CID 452110), DPPG2 (no Pubmed CID available, CAS 495403-05-9), dFdC (Pubmed CID: 60749).

The authors alone are responsible for the content and writing of the paper.

Supplementary material

11095_2014_1322_MOESM1_ESM.doc (404 kb)
ESM 1 (DOC 404 kb)

References

  1. 1.
    Heinemann V. Gemcitabine: progress in the treatment of pancreatic cancer. Oncology. 2001;60(1):8–18.PubMedCrossRefGoogle Scholar
  2. 2.
    Toschi L, Finocchiaro G, Bartolini S, Gioia V, Cappuzzo F. Role of gemcitabine in cancer therapy. Future Oncol. 2005;1(1):7–17.PubMedCrossRefGoogle Scholar
  3. 3.
    Hensley ML, Maki R, Venkatraman E, Geller G, Lovegren M, Aghajanian C, et al. Gemcitabine and docetaxel in patients with unresectable leiomyosarcoma: results of a phase II trial. J Clin Oncol. 2002;20(12):2824–31.PubMedCrossRefGoogle Scholar
  4. 4.
    Maki RG, Wathen JK, Patel SR, Priebat DA, Okuno SH, Samuels B, et al. Randomized phase II study of gemcitabine and docetaxel compared with gemcitabine alone in patients with metastatic soft tissue sarcomas: results of sarcoma alliance for research through collaboration study 002. J Clin Oncol. 2007;25(19):2755–63.PubMedCrossRefGoogle Scholar
  5. 5.
    Garcia-Del-Muro X, Lopez-Pousa A, Maurel J, Martin J, Martinez-Trufero J, Casado A, et al. Randomized phase II study comparing gemcitabine plus dacarbazine versus dacarbazine alone in patients with previously treated soft tissue sarcoma: a Spanish Group for Research on Sarcomas study. J Clin Oncol. 2011;29(18):2528–33.PubMedCrossRefGoogle Scholar
  6. 6.
    Movva S, Verschraegen C. Systemic management strategies for metastatic soft tissue sarcoma. Drugs. 2011;71(16):2115–29.PubMedCrossRefGoogle Scholar
  7. 7.
    Mini E, Nobili S, Caciagli B, Landini I, Mazzei T. Cellular pharmacology of gemcitabine. Ann Oncol. 2006;17 suppl 5:v7–12.PubMedCrossRefGoogle Scholar
  8. 8.
    Bornmann C, Graeser R, Esser N, Ziroli V, Jantscheff P, Keck T, et al. A new liposomal formulation of Gemcitabine is active in an orthotopic mouse model of pancreatic cancer accessible to bioluminescence imaging. Cancer Chemother Pharmacol. 2008;61(3):395–405.PubMedCrossRefGoogle Scholar
  9. 9.
    Jantscheff P, Esser N, Graeser R, Ziroli V, Kluth J, Unger C, et al. Liposomal gemcitabine (GemLip)-efficient drug against hormone-refractory Du145 and PC-3 prostate cancer xenografts. Prostate. 2009;69(11):1151–63.PubMedCrossRefGoogle Scholar
  10. 10.
    Maeda H. The enhanced permeability and retention (EPR) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting. Adv Enzyme Regul. 2001;41:189–207.PubMedCrossRefGoogle Scholar
  11. 11.
    Judson I, Radford JA, Harris M, Blay JY, van Hoesel Q, Le Cesne A, et al. Randomised phase II trial of pegylated liposomal doxorubicin (DOXIL/CAELYX) versus doxorubicin in the treatment of advanced or metastatic soft tissue sarcoma: a study by the EORTC Soft Tissue and Bone Sarcoma Group. Eur J Cancer. 2001;37(7):870–7.PubMedCrossRefGoogle Scholar
  12. 12.
    Harrington KJ, Mohammadtaghi S, Uster PS, Glass D, Peters AM, Vile RG, et al. Effective targeting of solid tumors in patients with locally advanced cancers by radiolabeled pegylated liposomes. Clin Cancer Res. 2001;7(2):243–54.PubMedGoogle Scholar
  13. 13.
    Laginha KM, Verwoert S, Charrois GJ, Allen TM. Determination of doxorubicin levels in whole tumor and tumor nuclei in murine breast cancer tumors. Clin Cancer Res. 2005;11(19 Pt 1):6944–9.PubMedCrossRefGoogle Scholar
  14. 14.
    Seynhaeve ALB, Dicheva BM, Hoving S, Koning GA, ten Hagen TLM. Intact Doxil is taken up intracellularly and released doxorubicin sequesters in the lysosome: Evaluated by in vitro/in vivo live cell imaging. J Control Release. 2013;172(1):330–40.PubMedCrossRefGoogle Scholar
  15. 15.
    Lindner LH, Hossann M. Factors affecting drug release from liposomes. Curr Opin Drug Discov Devel. 2010;13(1):111–23.PubMedGoogle Scholar
  16. 16.
    Landon CD, Park J, Needham D, Dewhirst MW. Nanoscale drug delivery and hyperthermia: the materials design and preclinical and clinical testing of low temperature-sensitive liposomes used in combination with mild hyperthermia in the treatment of local cancer. Open Nanomed J. 2011;3:38–64.PubMedCrossRefPubMedCentralGoogle Scholar
  17. 17.
    Grüll H, Langereis S. Hyperthermia-triggered drug delivery from temperature-sensitive liposomes using MRI-guided high intensity focused ultrasound: drug delivery research in Europe. J Control Release. 2012;161(2):317–27.PubMedCrossRefGoogle Scholar
  18. 18.
    May JP, Li S. Hyperthermia-induced drug targeting. Expert Opin Drug Deliv. 2013;10(4):511–27.PubMedCrossRefGoogle Scholar
  19. 19.
    Manzoor AA, Lindner LH, Landon CD, Park J, Simnick AJ, Dreher MR, et al. Overcoming limitations in nanoparticle drug delivery: triggered, intravascular release to improve drug penetration into tumors. Cancer Res. 2012;72(21):5566–75.PubMedCrossRefPubMedCentralGoogle Scholar
  20. 20.
    Yatvin MB, Weinstein JN, Dennis WH, Blumenthal R. Design of liposomes for enhanced local release of drugs by hyperthermia. Science. 1978;202(4374):1290–3.PubMedCrossRefGoogle Scholar
  21. 21.
    Chelvi TP, Ralhan R. Designing of thermosensitive liposomes from natural lipids for multimodality cancer therapy. Int J Hyperthermia. 1995;11(5):685–95.PubMedCrossRefGoogle Scholar
  22. 22.
    Chelvi TP, Ralhan R. Hyperthermia potentiates antitumor effect of thermosensitive-liposome-encapsulated melphalan and radiation in murine melanoma. Tumour Biol. 1997;18(4):250–60.PubMedCrossRefGoogle Scholar
  23. 23.
    Hosokawa T, Sami M, Kato Y, Hayakawa E. Alteration in the temperature-dependent content release property of thermosensitive liposomes in plasma. Chem Pharm Bull (Tokyo). 2003;51(11):1227–32.CrossRefGoogle Scholar
  24. 24.
    Lindner LH, Hossann M, Vogeser M, Teichert N, Wachholz K, Eibl H, et al. Dual role of hexadecylphosphocholine (miltefosine) in thermosensitive liposomes: active ingredient and mediator of drug release. J Control Release. 2008;125(2):112–20.PubMedCrossRefGoogle Scholar
  25. 25.
    Woo J, Chiu GN, Karlsson G, Wasan E, Ickenstein L, Edwards K, et al. Use of a passive equilibration methodology to encapsulate cisplatin into preformed thermosensitive liposomes. Int J Pharm. 2008;349(1–2):38–46.PubMedCrossRefGoogle Scholar
  26. 26.
    Hossann M, Wiggenhorn M, Schwerdt A, Wachholz K, Teichert N, Eibl H, et al. In vitro stability and content release properties of phosphatidylglyceroglycerol containing thermosensitive liposomes. Biochim Biophys Acta. 2007;1768(10):2491–9.PubMedCrossRefGoogle Scholar
  27. 27.
    Hossann M, Wang T, Wiggenhorn M, Schmidt R, Zengerle A, Winter G, et al. Size of thermosensitive liposomes influences content release. J Control Release. 2010;147(3):436–43.PubMedCrossRefGoogle Scholar
  28. 28.
    Lindner LH, Eichhorn ME, Eibl H, Teichert N, Schmitt-Sody M, Issels RD, et al. Novel temperature-sensitive liposomes with prolonged circulation time. Clin Cancer Res. 2004;10(6):2168–78.PubMedCrossRefGoogle Scholar
  29. 29.
    Eibl H. Synthesis of glycerophospholipids. Chem Phys Lipids. 1980;26(4):405–29.PubMedCrossRefGoogle Scholar
  30. 30.
    Eibl H, Lands WE. A new, sensitive determination of phosphate. Anal Biochem. 1969;30(1):51–7.PubMedCrossRefGoogle Scholar
  31. 31.
    Lanz C, Früh M, Thormann W, Cerny T, Lauterburg BH. Rapid determination of gemcitabine in plasma and serum using reversed-phase HPLC. J Sep Sci. 2007;30(12):1811–20.PubMedCrossRefGoogle Scholar
  32. 32.
    Hossann M, Syunyaeva Z, Schmidt R, Zengerle A, Eibl H, Issels RD, et al. Proteins and cholesterol lipid vesicles are mediators of drug release from thermosensitive liposomes. J Control Release. 2012;162(2):400–6.PubMedCrossRefGoogle Scholar
  33. 33.
    Massing U, Cicko S, Ziroli V. Dual asymmetric centrifugation (DAC)–a new technique for liposome preparation. J Control Release. 2008;125(1):16–24.PubMedCrossRefGoogle Scholar
  34. 34.
    Grit M, Crommelin DJ. Chemical stability of liposomes: implications for their physical stability. Chem Phys Lipids. 1993;64(1–3):3–18.PubMedCrossRefGoogle Scholar
  35. 35.
    Moog R, Brandl M, Schubert R, Unger C, Massing U. Effect of nucleoside analogues and oligonucleotides on hydrolysis of liposomal phospholipids. Int J Pharm. 2000;206(1–2):43–53.PubMedCrossRefGoogle Scholar
  36. 36.
    Haveman J, Rietbroek RC, Geerdink A, van Rijn J, Bakker PJ. Effect of hyperthermia on the cytotoxicity of 2′,2′-difluorodeoxycytidine (gemcitabine) in cultured SW1573 cells. Int J Cancer. 1995;62(5):627–30.PubMedCrossRefGoogle Scholar
  37. 37.
    van Bree C, Beumer C, Rodermond HM, Haveman J, Bakker PJ. Effectiveness of 2′,2′difluorodeoxycytidine (Gemcitabine) combined with hyperthermia in rat R-1 rhabdomyosarcoma in vitro and in vivo. Int J Hyperthermia. 1999;15(6):549–56.PubMedCrossRefGoogle Scholar
  38. 38.
    Li L, ten Hagen TLM, Bolkestein M, Gasselhuber A, Yatvin J, van Rhoon GC, et al. Improved intratumoral nanoparticle extravasation and penetration by mild hyperthermia. J Control Release. 2013;167(2):130–7.PubMedCrossRefGoogle Scholar
  39. 39.
    Moog R, Burger AM, Brandl M, Schuler J, Schubert R, Unger C, et al. Change in pharmacokinetic and pharmacodynamic behavior of gemcitabine in human tumor xenografts upon entrapment in vesicular phospholipid gels. Cancer Chemother Pharmacol. 2002;49(5):356–66.PubMedCrossRefGoogle Scholar
  40. 40.
    Tempero M, Plunkett W, van Ruiz HV, Hainsworth J, Hochster H, Lenzi R, et al. Randomized phase II comparison of dose-intense gemcitabine: thirty-minute infusion and fixed dose rate infusion in patients with pancreatic adenocarcinoma. J Clin Oncol. 2003;21(18):3402–8.PubMedCrossRefGoogle Scholar
  41. 41.
    Hughes TL, Hahn TM, Reynolds KK, Shewach DS. Kinetic analysis of human deoxycytidine kinase with the true phosphate donor uridine triphosphate. Biochemistry. 1997;36(24):7540–7.PubMedCrossRefGoogle Scholar
  42. 42.
    Grunewald R, Kantarjian H, Keating MJ, Abbruzzese J, Tarassoff P, Plunkett W. Pharmacologically directed design of the dose rate and schedule of 2′,2′-difluorodeoxycytidine (Gemcitabine) administration in leukemia. Cancer Res. 1990;50(21):6823–6.PubMedGoogle Scholar
  43. 43.
    Abbruzzese JL, Grunewald R, Weeks EA, Gravel D, Adams T, Nowak B, et al. A phase I clinical, plasma, and cellular pharmacology study of gemcitabine. J Clin Oncol. 1991;9(3):491–8.PubMedGoogle Scholar
  44. 44.
    Hallett FR, Marsh J, Nickel BG, Wood JM. Mechanical properties of vesicles. II. A model for osmotic swelling and lysis. Biophys J. 1993;64(2):435–42.PubMedCrossRefPubMedCentralGoogle Scholar
  45. 45.
    Hijnen NM, Heijman E, Kohler MO, Ylihautala M, Ehnholm GJ, Simonetti AW, et al. Tumour hyperthermia and ablation in rats using a clinical MR-HIFU system equipped with a dedicated small animal set-up. Int J Hyperthermia. 2012;28(2):141–55.PubMedCrossRefGoogle Scholar
  46. 46.
    Hatzakis NS, Bhatia VK, Larsen J, Madsen KL, Bolinger P, Kunding AH, et al. How curved membranes recruit amphipathic helices and protein anchoring motifs. Nat Chem Biol. 2009;5(11):835–41.PubMedCrossRefGoogle Scholar
  47. 47.
    Koning GA, Eggermont AMM, Lindner LH, ten Hagen TLM. Hyperthermia and thermosensitive liposomes for improved delivery of chemotherapeutic drugs to solid tumors. Pharm Res. 2010;27(8):1750–4.PubMedCrossRefPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Simone Limmer
    • 1
    • 2
  • Jasmin Hahn
    • 1
  • Rebecca Schmidt
    • 1
  • Kirsten Wachholz
    • 1
  • Anja Zengerle
    • 1
  • Katharina Lechner
    • 1
    • 2
  • Hansjörg Eibl
    • 3
  • Rolf D. Issels
    • 1
    • 2
  • Martin Hossann
    • 1
    • 2
    • 4
  • Lars H. Lindner
    • 1
    • 2
  1. 1.Department of Internal Medicine IIIUniversity Hospital Munich Ludwig-Maximilians UniversityMunichGermany
  2. 2.CCG Tumor Therapy through Hyperthermia, Helmholtz Zentrum MünchenGerman Research Center for Environmental HealthMunichGermany
  3. 3.Max Planck Institute for Biophysical ChemistryGoettingenGermany
  4. 4.Medizinische Klinik und Poliklinik IIIKlinikum der Universität MünchenMunichGermany

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