Metastasizing, Luciferase Transduced MAT­Lu Rat Prostate Cancer Models: Follow up of Bolus and Metronomic Therapy with Doxorubicin as Model Drug.

The most fatal outcomes of prostate carcinoma (PCa) result from hormone-refractory variants of the tumor, especially from metastatic spread rather than from primary tumor burden. The goal of the study was to establish and apply rat MAT-Lu prostate cancer tumor models for improved non-invasive live follow up of tumor growth and metastasis by in vivo bioluminescence. We established luciferase transduced MAT-Lu rat PCa cells and studied tumor growth and metastatic processes in an ectopic as well as orthotopic setting. An intravenous bolus treatment with doxorubicin was used to demonstrate the basic applicability of in vivo imaging to follow up therapeutic intervention in these models. In vitro analysis of tissue homogenates confirmed major metastatic spread of subcutaneous tumors into the lung. Our sensitive method, however, for the first time detects metastasis also in lymph node (11/24), spleen (3/24), kidney (4/24), liver (5/24), and bone tissue (femur or spinal cord - 5/20 and 12/20, respectively). Preliminary data of orthotopic implantation (three animals) showed metastatic invasion to investigated organs in all animals but with varying preference (e.g., to lymph nodes). Intravenous bolus treatment of MAT-Lu PCa with doxorubicin reduced subcutaneous tumor growth by about 50% and the number of animals affected by metastatic lesions in lymph nodes (0/4), lung (3/6) or lumbar spine (0/2), as determined by in vivo imaging and in vitro analysis. Additionally, the possible applicability of the luciferase transduced MAT-Lu model(s) to study basic principles of metronomic therapies via jugular vein catheter, using newly established active microport pumping systems, is presented.

Anti-metastatic effects of liposomal gemcitabine in a human orthotopic LNCaP prostate cancer xenograft model.

Fatal outcomes of prostate carcinoma (PCa) mostly result from metastatic spread rather than from primary tumor burden. Here, we monitored growth and metastatic spread of an orthotopic luciferase/GFP-expressing LNCaP PCa xenograft model in SCID mice by in vivo imaging and in vitro luciferase assay of tissues homogenates. Although the metastatic spread generally shows a significant correlation to primary tumor volumes, the susceptibility of various tissues to metastatic invasion was different in the number of affected animals as well as in absolute metastatic burden in the individual tissues. Using this xenograft model we showed that treatment with liposomal gemcitabine (GemLip) inhibited growth of the primary tumors (83.9 +/- 6.4%; P = 0.009) as well as metastatic burden in lymph nodes (95.6 +/- 24.0%; P = 0.047), lung (86.5 +/- 10.5%; P = 0.015), kidney (88.4 +/- 9.2%; P = 0.045) and stomach (79.5 +/- 6.6%; P = 0.036) already at very low efficient concentrations (8 mg/kg) as compared to conventional gemcitabine (360 mg/kg). Our data show that this orthotopic LNCaP xenograft PCa model seems to reflect the clinical situation characterized by the fact that at time of diagnosis, prostate neoplasms are biologically heterogeneous and thus, it is a useful model to investigate new anti-metastatic therapies.

Liposomal gemcitabine (GemLip)-efficient drug against hormone-refractory Du145 and PC-3 prostate cancer xenografts.

BACKGROUND: Gemcitabine (Gemc) is an efficient chemotherapeutic drug in various cancer types (e.g., pancreas) but has only limited effects on hormone-refractory prostate cancer (HRPCa). Since HRPCa cells are highly sensitive to even low doses of Gemc in vitro, the lack of clinical effects might be due to rapid degradation of Gemc by deaminases combined with impaired accumulation in tumor tissue and PCa cells. Liposomal formulation (GemLip) is expected to protect the entrapped cytotoxic substance from enzymatic degradation and furthermore augment its accumulation within tumor tissues due to an enhanced permeability of the tumor vessels. METHODS: Anti-tumoral and anti-metastatic activity of GemLip and Gemc were investigated in two luciferase-expressing, human hormone-refractory PC-3 and Du145 HRPCa xenograft models in immunodeficient mice. Tumor growth was monitored by in vivo luminescence imaging (orthotopic) or callipering (subcutaneous). Anti-metastatic effects of treatment were determined by in vitro luciferase assay of the tissues. RESULTS: Tumor growth of subcutaneous Du145 xenografts was significantly inhibited only by GemLip (8 mg/kg: P = 0.014 and 6 mg/kg: P = 0.011) but not by conventional Gemc (360 mg/kg). In contrast, growth of orthotopic PC-3 xenografts was significantly inhibited by both, GemLip (P = 0.041) and Gemc (P = 0.002). The drugs furthermore strongly reduced spleen and liver metastases in this model. CONCLUSIONS: As shown by the very low efficient concentration of GemLip, liposomal entrapment of Gemc greatly enhances its activity. GemLip has, even at very low doses, a significant anti-tumoral and anti-metastatic therapeutic effect in HRPCa xenografts in vivo and was beneficial even when the conventional Gemc failed.

Antimetastatic effects of liposomal gemcitabine and empty liposomes in an orthotopic mouse model of pancreatic cancer.

OBJECTIVES: Test the efficacy of liposomal gemcitabine (GemLip) on primary tumor and metastases using the pancreatic tumor cell line AsPC1 implanted orthotopically into nude mice. METHODS: The efficacy of gemcitabine and GemLip cells was tested on luciferase-transduced AsPC1 cells in vitro as well as implanted orthotopically into the pancreata of nude mice. RESULTS: In vitro, the IC50s for GemLip and gemcitabine were 20 nM and 140 nM, respectively. However, when applied against established tumors, GemLip (8 mg/kg) blocked tumor growth for 5 consecutive weeks according to bioluminescence measurements in vivo. Gemcitabine (240 mg/kg) had no effect on luciferase-monitored tumor growth. When analyzed at the time of necropsy, GemLip strongly reduced tumor size (-64% +/- [SD] 27%; ***P < 0.0001), whereas gemcitabine only weakly (-36% +/- 37%) affected tumor size. Empty liposomes had no effect on the tumor size. GemLip and empty liposomes both significantly interfered with the metastatic spread to the liver, as measured using luciferase assays (GemLip, *P = 0.01; empty liposomes, *P = 0.036). In addition, they showed effects against spleen, as well as peritoneal metastases. CONCLUSIONS: GemLip presents an effective new formulation of gemcitabine, combining the targeting and protective features of the liposomes with their antimetastatic effects to target pancreatic cancer.

Preparation of small amounts of sterile siRNA-liposomes with high entrapping efficiency by dual asymmetric centrifugation (DAC).

Liposomal formulation of siRNA is an attractive approach for improving its delivery in vivo, shielding the RNA from nucleases and promoting tumor targeting. Here, the production of very small batch sizes of siRNA-liposomes by using the "dual asymmetric centrifugation (DAC)" technique was investigated. This new technique combines rapid and sterile liposome preparation with very high entrapping efficiencies. DAC is here presented in conjunction with a non-destructive microscale analysis based on double fluorescence labeling, which enables monitoring of siRNA integrity during the liposomal preparation. Integrity is reflected in spatial proximity of the dyes, which results in measurable fluorescence resonance energy transfer (FRET). The combination of DAC and the sensitive FRET analysis allows the handling of batch sizes down to 20 mg of conventional liposomes (CL) and sterically stabilized liposomes (SL). These were prepared in common 2 ml reaction tubes and loaded with calcein or labeled siRNA. Liposome sizes were 79+/-16 nm for CL and 109+/-9 nm for SL loaded with siRNA. Trapping efficiencies ranged from 43 to 81%, depending on batch size, enclosed compound, and liposome composition. FRET monitoring showed that the siRNA remained intact throughout DAC and that liposomal formulations protected the siRNA from nucleases. siRNA-liposomes remained stable for at least 3 months.

Dual asymmetric centrifugation (DAC)--a new technique for liposome preparation.

This is the first report on the use of a "dual asymmetric centrifuge (DAC)" for preparing liposomes. DAC differs from conventional centrifugation by an additional rotation of the sample around its own vertical axis: While the conventional centrifugation constantly pushes the sample material outwards, this additional rotation constantly forces the sample material towards the center of the centrifuge. This unique combination of two contra rotating movements results in shear forces and thus, in efficient homogenization. We demonstrated that it is possible to prepare liposomes by DAC, by homogenizing a rather concentrated blend of hydrogenated phosphatidylcholine and cholesterol (55:45 mol%) and 0.9% NaCl-solution, which results in a viscous vesicular phospholipid gel (VPG). The resulting VPG can subsequently be diluted to a conventional liposome dispersion. Since DAC is intended to make sterile preparations of liposomes, or to entrap toxic/radioactive compounds, the process was performed within a sealed vial. It could be shown that the DAC speed, the lipid concentration, the homogenization time and the addition of a mixing aid (glass beads) are all critical for the size of the liposomes. Optimized conditions resulted in liposomes of 60+/-5 nm and a trapping efficacy of 56+/-3.3% for the model compound calcein.

PAC fixed dose: pharmacokinetics of a 1-hour paclitaxel infusion and comparison to BSA-normalized drug dosing.

BACKGROUND: The aim of this study was to determine the pharmacokinetics (PKs) of a 175-mg fixed dose of paclitaxel (PAC) after a 1-h infusion in cancer patients and to compare them with the PK parameters from a study with a dose normalized to the body surface area (BSA) (100 mg/m2). PATIENTS AND METHODS: PAC PKs were studied during the first course of therapy in 13 patients. A fixed dose of 175 mg PAC was administered weekly by a 1-h infusion to patients with advanced cancer. Total PAC in serum was quantified by high-performance liquid chromatography (HPLC). PK parameters were calculated by non-compartmental and model-dependent methods. RESULTS: The mean BSA of 12 patients (1 patient excluded from all analyses because of prolonged infusion duration) was 1.79 m2 (coefficient of variation (CV) 7.8%), the mean dose referred to the individual BSAs was 98.3 mg/m2 (CV 8.3%). The mean area under the curve (AUC) was 6,193 ng/ml x h (CV 46%), the mean plasma clearance (Clp) was 19.7 l/h/m2 (CV 45%), and the volume of distribution at steady state (Vss) was 121.6 l/m2 (CV 52%). The mean residence time (MRT) was 7.6 h (CV 46%), the mean distribution half-life (t1/2 alpha) of PAC(tot) was 0.4 h (CV 62%), and the elimination half-life (t1/2 beta) 10.0 h (CV 42%). Maximum plasma concentration Cmax was 3,161 ng/ml (CV 36%). The mean time above 0.05 microM (42.7 ng/ml) was 19.7 h, and the mean time above 0.1 microM (85.4 ng/ml) was 10.6 h. CONCLUSIONS: In this study, a fixed dose of PAC of 175 mg corresponds to a mean BSA-normalized dose of 98.3 mg/m2 (range 88.8-117.4 mg/m2). A higher variability of PK parameters was observed compared to previously published results of a PK study with BSA-normalized dosing of 100 mg/m2. However, the AUC and the time above threshold concentrations did not depend on the dose. Therefore, a fixed dose of 175 mg weekly could be an option for palliative treatment with PAC and may offer a simple but effective schedule for PAC treatment.

Quantification of various phosphatidylcholines in liposomes by enzymatic assay.

The purpose of this research was to adapt a colorimetric, phospholipase D-based serum-phospholipid assay for the quantification of phosphatidylcholine (PC) in liposomes using a microtitre plate reader. PC from natural egg PC liposomes was quantified reliably. In contrast, poor sensitivity was found for liposomes composed of saturated PCs (di-palmitoyl-phosphatidylcholine [DPPC], hydrogenated egg PC). Triton X-100 was then added to the liposomes followed by heating above the phase transition temperature. This modified sample preparation resulted in recoveries of 102.6% +/- 1.0%, 104.4% +/- 7.6%, and 109.4% +/- 3.2% for E80, E80-3/cholesterol, and DPPC liposomes, respectively. Absolute quantification of unknown PCs against a choline chloride standard is feasible, but relative measurements against the very same PC are recommended whenever possible. Validation experiments revealed an absolute quantification limit of 1.25 microg per assay, a good linearity in the range of 25 to 1000 microg/mL PC (r2> or = 0.9990) and a quite high accuracy (99.8%-101.4% of theory) and precision (relative standard deviation < or = 3.2%) for all 3 PCs studied. The method is thus regarded as suitable for sensitive, rapid, and reliable routine quantification of PCs in liposomes.