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Pharmacodynamic Behavior of Liposomal Antisense Oligonucleotides Targeting Her-2/neu and Vascular Endothelial Growth Factor in an Ascitic MDA435/LCC6 Human Breast Cancer Model

Pharmacodynamic Behavior of Liposomal Antisense Oligonucleotides Targeting Her-2/neu and Vascular... [Cancer Biology & Therapy 3:2, 197-204, February 2004]; ©2004 Landes Bioscience Research Papers Pharmacodynamic Behavior of Liposomal Antisense Oligonucleotides Targeting Her-2/neu and Vascular Endothelial Growth Factor in an Ascitic MDA435/LCC6 Human Breast Cancer Model 1, ABSTRACT Dawn N. Waterhouse * Wieslawa H. Dragowska The nature of anti-cancer therapeutics is currently undergoing a paradigm change, with biologic agents slowly being introduced into the therapeutic armory, displacing or Karen A. Gelmon complimenting the traditionally used cytotoxic agents. These new agents include mono- 1,2 Lawrence D. Mayer clonal antibodies, recombinant DNA, antisense oligonucleotides (ASO) and others. To 1,3 Marcel B. Bally assess the new therapeutics, new predictive models are required. Utilizing the MDA435/LCC6 human breast cancer xenograft model, the pharmacokinetic behavior of British Columbia Cancer Research Centre; Department of Advanced Therapeutics; antisense oligonucleotides targeted against vascular endothelial growth factor and Vancouver, B.C., Canada HER-2/neu was assessed. For pharmacodynamic analysis, ASO in buffer or encapsulated University of British Columbia; Faculty of Pharmaceutical Sciences; Vancouver, B.C., in a liposomal formulation were injected intravenously or intraperitoneally into Canada MDA435/LCC6 ascites tumor-bearing mice. Plasma antisense elimination, tissue distri- University of British Columbia; Department of Pathology and Laboratory Medicine; bution, total peritoneal antisense and peritoneal cell associated antisense levels were Vancouver, B.C., Canada determined. Liposomal encapsulation led to significant decreases in the plasma elimination *Correspondence to: Dawn N. Waterhouse; British Columbia Cancer Research rate, as evidenced by an approximate 10-fold increase in mean AUC over 24 hours, as Centre; Department of Advanced Therapeutics; 601 West 10th Avenue; Vancouver, well as enhanced peritoneal cell delivery in mice bearing ascites tumors. Tissue distribution B.C., Canada; Tel.: 604.877.6011; Fax: 604.877.6011; Email: dwater@bccancer.bc.ca studies of both free and liposome encapsulated ASO indicated that ASO distribution was Received 09/16/03; Accepted 09/16/03 dictated primarily by the liposomal carrier when administered in liposomal form. Previously published online as a Cancer Biology & Therapy E-publication: http://www.landesbioscience.com/journals/cbt/abstract.php?id=622 INTRODUCTION KEY WORDS In North America and Europe, approximately 11% of all women, and 1% of all men pharmacokinetic, pharmacodynamic, antisense will develop breast cancer. Many of these patients will also develop metastatic disease, with oligonucleotide, liposome, xenograft tumor approximately 35% of those who develop breast cancer ultimately succumbing to their disease. For this reason, there is an obvious need for developing more efficacious treatment ABBREVIATIONS strategies. Research has elucidated several specific prognostic and predictive factors to ASO antisense oligonucleotides identify patients at high risk for more aggressive disease, metastasis and for recurrence of AUCarea under the curve disease in order to combat these statistics. Several studies have demonstrated, for example, CHE cholesteryl hexadecyl ether that an increase in cytosolic levels of vascular endothelial growth factor (VEGF) in tumor Chol cholesterol tissue samples is indicative of poorer prognosis for patients with node-negative breast DEAE diethylaminoethyl 1,2 3 carcinoma. VEGF plays a key role in the process of angiogenesis, and the extent of DMEM Dulbecco’s minimal essential 4-8 tumor angiogenesis is now widely accepted as a prognostic factor in several cancer types, medium DODAP dioleoyl dimethylammonium with higher levels being indicative of more aggressive disease. propane We believe that anti-VEGF treatment modalities will be of great interest in the context FBS fetal bovine serum of the drug combination treatment strategies being developed for treatment of molecular- H2A-ASO anti-HER-2/neu antisense ly defined aggressive disease. Investigators have demonstrated a link between VEGF and HBS HEPES Buffered saline HER-2/neu, another marker of poor prognosis in both node positive and node negative HBSS Hank’s balanced salt solution 9-14 breast cancers. Over-expression of HER-2/neu is found in 20–25% of breast cancer modified 15-18 patients, and is predictive of shorter disease free and overall survival. Research is also PEG polyethylene glycol elucidating a benefit from targeting both VEGF and HER-2/neu in conjunction in the PEG-C14CER PEG2000C14ceramide therapeutic intervention of breast cancer. VEGF vascular endothelial growth The discovery of new prognostic and predictive markers for breast cancer is obviously factor VEGF-ASO anti-VEGF antisense of great potential therapeutic value, however in order to exploit these markers in the devel- opment of treatment strategies, researchers require appropriate in vivo models. Thus, when ACKNOWLEDGEMENTS considering strategies targeting HER-2/neu and VEGF, the cell lines used to establish tumor models must also express the targets. The development of a human xenograft ascites This work was funded by a National Cancer model fulfilling some of these requirements has been achieved in the MDA435/LCC6 Institute of Canada, Breast Cancer Initiative grant. model. This model has a reproducible growth curve in immune compromised mice and D.N.W. is a Canadian Breast Cancer Foundation exhibits sensitivity to cytotoxic drugs known to be active in the treatment of human breast Research Fellow. cancer patients. The cells are easily maintained in vitro, thus allowing rapid assessment of drugs both in vitro and in vivo. In this report we provide a further characterization of the MDA435/LCC6 model for VEGF and HER-2/neu expression. www.landesbioscience.com Cancer Biology & Therapy 197 © Landes Bioscience 2004. Not for distribution. ANTISENSE PK IN THE MDA435/LCC6 BREAST CANCER MODEL The use of this model to assess the pharmacodynamic behavior of for antisense and 130 dpm/µg for lipid. Antisense and lipid solutions were briefly heated to 60˚C, then combined in a weight-to-weight ratio of 0.2 to two antisense oligonucleotide sequences designed to inhibit expres- 1.0, antisense being added drop-wise to lipids in ethanol while mixing sion of VEGF and HER-2/neu has been evaluated. ASOs have been vigorously. Mixtures were subjected to 5 freeze-thaw cycles, consisting of extensively researched as pharmaceutical agents, however even with immersion in liquid nitrogen followed by thawing at 65 C, prior to repeated the chemical modification of the phosphate backbone to a more (10X) extrusion through three stacked 100 nm pore size polycarbonate stable phosphorothioate bond, these ASO are rapidly eliminated membranes (Nucleopore, Pleasanton, CA) using a thermobarrel equipped from the circulation, primarily by the kidneys. Although ASOs are extrusion device (Northern Lipids Inc., Vancouver, BC). Following extrusion, 22-23 efficacious when given in vivo, activity is often dependent on samples were dialyzed against 300 mM citrate buffer, pH 4.0 for two hours use of aggressive dose scheduling involving daily injections for time to remove ethanol, then against pH 7.5 HEPES buffered saline (HBS) for a periods in excess of two weeks or use of micro-infusion pumps. minimum of 12 hours at 4 C. Liposomes were then passed over a diethy- Thus it may be argued that drug carrier formulations may engender laminoethyl (DEAE)-agarose anion exchange column (BioRad, Mississauga, Ontario, Canada) equilibrated in HBS to remove unencapsulated antisense. improved pharmacological properties including enhanced circula- The size of the resultant liposomes was typically about 130 nm as deter- tion longevity, as well as increased delivery of the ASO to sites where mined by analysis with a Nicomp 270 submicron particle sizer (Pacific the target cells are localized. The two antisense oligonucleotides Scientific, Santa Barbara, CA), operating at 632.8 nm. utilized in these studies were a 21-mer with a phosphorothioate As necessary, lipid and antisense concentrations were determined by backbone, targeted against human VEGF with demonstrated activity 14 3 [ C] and [ H] levels (respectively), by mixing samples with 5 mL of against production of this protein in an in vitro model, and a PicoFluor 40 scintillation cocktail (Packard, Groningen, The Netherlands), 15-mer phosphorothioate ASO specific for the area of HER-2/neu and counting with a Packard 1900 scintillation counter (Meriden, CT). 26-27 mRNA immediately upstream of the initiation codon. These Encapsulation of antisense was determined using scintillation counts or antisense sequences were encapsulated in a novel liposomal formula- by solubilization method as follows. A 10 µl aliquot of liposomal antisense tion, recently described by Semple et al. and the pharmacodynamic or water (for blank) was added to 240 µl distilled water. This was combined behavior of these ASOs, delivered in free or liposomal form, was with 750 µl CHCl /MeOH (1.0:2.1 vol:vol) and 100 µl MeOH. The mix- determined using the MDA435/LCC6 ascites model. tures were vortexed until clear, and absorbance determined at 260 nm. Concentration was determined according to the following calculation: Concentration = (A )(Extinction coefficient of antisense, ε)(dilution) MATERIALS AND METHODS Where ε = 34.5 µg/OD unit. In Vivo Studies. Female SCID/Rag2m mice (Taconic, Germantown, SK-BR-3, MCF7 and A431 cells were obtained from the American Type NY), n = 8, were injected i.p. with 2 x 10 MDA435/LCC6 cells, or s.c. Culture Collection (Rockville, MD). MDA-MB-435 cells were from the with 5 x 10 MDA435/LCC6 cells, in a total volume of 200 or 50 µL, tumor repository of the National Cancer Institute (Frederick, MD). respectively. These cell numbers give consistent and reproducible tumor MDA435/LCC6 cells were a generous gift from Dr. Robert Clarke, growth rates. Total volume of ascites fluid, ascites cell number, and ascites Georgetown University (Washington, DC). SK-BR-3 cells were maintained and serum VEGF levels were determined for the ascites model after 0, 7, 14, in McCoy’s 5A medium with 10% fetal bovine serum (FBS), MCF7 were 21 or 28 days. Immunohistochemical analysis was performed on sections maintained in RPMI 1640, 10% FBS and 2 mM L-glutamine. All other derived from solid tumors and these sections were stained for VEGF, cells were maintained in Dulbecco’s minimal essential medium (DMEM) HER-2/neu and bcl-2 proteins as described below. For determination of supplemented with 10% FBS and 2 mM L-glutamine. Cultures were sus- tained at 37 C and in an atmosphere of 5% CO . All media, Hank’s ascites VEGF levels, an enzyme linked immunosorbent assay (ELISA) (R&D Systems) was used, using standards for serum VEGF levels. To assess Balanced Salt Solution Modified (HBSS), and trypsin-EDTA were obtained from StemCell Technologies Inc. (Vancouver, BC, Canada). levels of VEGF in cells grown in culture, cells were plated in 25 cm tissue culture flasks. When cells were confluent, supernatant was collected and Phosphorothioate backbone anti-HER-2/neu antisense oligonucleotide (H2A-ASO) and anti-VEGF-A antisense oligonucleotide (VEGF-ASO) assayed by VEGF ELISA (Quantikine human VEGF quantitative colori- metric sandwich ELISA, R&D Systems, Minneapolis, MN). were synthesized by Hybridon Inc. (Cambridge, MA). Sequences of anti- sense oligonucleotides were, H2A-ASO: 5’-GGT GCT CAC TGC GGC Ascites fluid was collected by peritoneal lavage. For early time-points, a known volume of HBSS was injected into the peritoneum prior to lavage. and VEGF-ASO: 5’- AGA CAG CAG AAA GTT CAT GGT-3’, targeting the 15 bases immediately upstream of the initiation codon of human Cells were counted with a hemacytometer. For the s.c. model, tumor size was determined by the formula L(W )/2, with measurements being taken in HER-2/neu mRNA, and -3 to +18 of the human vascular endothelial 3 3 growth factor mRNA coding region, respectively. [ H]-VEGF-ASO and mm with digital calipers. This yielded tumor volume in mm , which we have determined to be roughly equivalent to tumor weight in grams. When [ H]-H2A-ASO were obtained from TriLink Biotechnologies, Inc. (San Diego, CA). [14C]-cholesteryl hexadecyl ether (CHE) was obtained from tumors reached a size of between 0.5 and 1 g, the animals were terminated, and tumors collected in formalin. Tumors were embedded in paraffin, and Amersham Canada Ltd. (Oakville, ON, Canada). All chemicals or reagents not otherwise mentioned were from Sigma (Oakville, ON, Canada). 5 µm sections prepared (Wax-It, Aldergrove, BC). Sections were stained for Preparation of Liposomal Antisense Oligonucleotides. Liposomal anti- VEGF using mouse anti-human VEGF monoclonal antibody, clone sense was prepared following the method of Semple et al. Stock lipid solu- 26503.11 from R&D Systems. Blocking agent and secondary antibody were tions were prepared in 100% ethanol. 1,2-distearoyl-sn-glycero-3-phospho- from the Vector Laboratories Vectastain Elite mouse ABC kit (Burlingame, choline (DSPC) (Northern Lipids Inc., Vancouver, BC), cholesterol (Chol) CA). Visualization was with ImmunoPure Metal Enhanced DAB (Sigma) and dioleoyl dimethylammonium propane (DODAP) (Avanti Polar Substrate kit (Pierce, Rockford, IL). Positive controls for VEGF staining Lipids, Alabaster, AL) were prepared at 20 mg/ml, while PEG2000 were sections of tumors derived following s.c. injection of 2 x 10 A431 C14Ceramide (PEG-C CER)(Northern Lipids Inc.) was prepared at 50 cells. mg/ml. Lipids were combined in a molar ratio of 2.0:2.5:4.5:1.0 Immunohistochemical staining for HER-2/neu was performed using an 14 30 (DODAP:DSPC:Chol:PEG-C -CER). As required, [ C]-CHE was added antibody cocktail, as described by Paik et al. Primary antibodies were poly- to the dissolved lipids as a nonexchangeable, nonmetabolizable marker. clonal rabbit anti-c-erbB-2 and monoclonal mouse anti-HER2 (c-erbB-2) Antisense was dissolved in 300 mM citrate buffer, pH 4.0 at a concentration antibody (Zymed Laboratories). Secondary antibodies and blocking solu- 3 3 of 3.33 mg/ml, with the addition of sufficient [ H]-H2A-ASO or [ H]- tions were an equal volume mixture from the Vector Laboratories VEGF-ASO for quantitation. Typical specific activities were 900 dpm/µg Vectastain Elite mouse and rabbit ABC kits. Slides were counterstained 198 Cancer Biology & Therapy 2004; Vol. 3 Issue 2 © Landes Bioscience 2004. Not for distribution. ANTISENSE PK IN THE MDA435/LCC6 BREAST CANCER MODEL Figure 1. (A) In vitro VEGF levels in supernatant collected from confluent MDA-MB-435 and MDA435/LCC6 cells following 24 hours incubation of matched cell number, measured by ELISA. Results in pg/ml ± standard error of the mean, and (B) in vivo VEGF levels from peritoneal fluid of MDA435/LCC6 derived ascites tumors (female SCID/Rag2m mice), collect- Figure 2. Immunohistochemical assessment of in vivo VEGF and HER-2/neu ed 0, 7, 14, 21 or 28 days post tumor cell inoculation. Insert shows serum levels. 5 x 10 A431 (A), SK-BR-3 (B), MDA-MB-435 (C, D) or VEGF concentration from these same animals. Ascites and serum were MDA435/LCC6 (E, F) were inoculated s.c. into female SCID/Rag2 mice. assayed by ELISA; results show pg and pg/ml, respectively, ± standard error Tumors were collected in formalin before reaching 1 gram in size, paraffin of the mean of 4 animals. embedded and stained immunohistochemically for VEGF (A, C and E) or HER-2/neu (B, D and F). Panels (A) and (B) are positive controls for VEGF and with Gill’s hematoxylin, dehydrated in ethanol, cleared in xylene and HER-2/neu staining, respectively. Visualization is with diaminobenzidine, mounted. Positive control sections for HER-2/neu staining were sections of where brown areas indicate positive staining. Tissues were counterstained tumors derived following s.c. injection of SK-BR-3 cells. with Gill’s hematoxylin. To determine the circulation longevity of the liposome encapsulated antisense in comparison to free antisense, groups of four female SCID/Rag2 counting. The liver, spleen, lung, kidney, heart and muscle were collected mice were injected i.v. (200 µl) or i.p. (500 µl) with either free or liposomal and weighed. Tissues (with the exception of the spleen) were made into a antisense at an antisense dose of 10 mg/kg. Samples were prepared with 30% homogenate in water using a Polytron tissue homogenizer. Two 3 3 14 [ H]-VEGF-ASO or [ H]-H2A-ASO and [ C]-CHE lipid label for quan- hundred microliters of this homogenate was transferred to scintillation vials. titation. Blood was collected by tail nick at 1 and 4 hours, and by cardiac Spleens were collected directly into scintillation vials. Solvable (500 µl) puncture at 24 hours. Serum antisense and liposomal lipid levels were was added to each vial, which were then incubated at 50°C overnight. The assessed by radiometric assay. This experiment was repeated in tumor bear- samples were cooled to room temperature prior to addition of 50 µL 200 ing mice. Female SCID/Rag2m mice were injected i.p. on day 0 with 2 x mM EDTA, 25 µl 10 M HCl and 200 µL 30% H O . This mixture was 2 2 10 MDA435/LCC6 cells. On day 17, free or liposomal antisense was incubated at room temperature for one hour prior to addition of 5 mL 3 3 14 injected. In these studies the mice were injected i.v. with [ H]-VEGF-ASO scintillation cocktail. Samples were analyzed for [ H] and [ C] by scintilla- 3 14 or [ H]-H2A-ASO and [ C]-CHE labeled liposomal antisense, at an anti- tion counting, and values corrected for tissue blood content. sense concentration of 10 mg/kg. At 1, 2, 4, 24 and 48 hours, groups of 4 Statistical analysis was performed with ANOVA one way post-hoc mice were sacrificed. Blood was collected by cardiac puncture. Peritoneal comparisons. lavages were performed by first injecting 3 ml HBSS into the peritoneal cavity and the abdomen was gently massaged for a period of 30 to 60 seconds. Subsequently the peritoneal fluid, with associated cells was carefully collected into sterile polystyrene tubes. Total volume was estimated and cell number determined using a hemocytometer. Aliquots (1 ml) were taken for scintillation www.landesbioscience.com Cancer Biology & Therapy 199 © Landes Bioscience 2004. Not for distribution. ANTISENSE PK IN THE MDA435/LCC6 BREAST CANCER MODEL a decline in the number of viable tumor cells in the peritoneum (data not shown) and this decrease in cell number correlated A B with the decline in peritoneal VEGF levels. As indicated in the insert to Figure 1B, VEGF levels in the serum of these mice steadily increased over the time period of tumor growth, with VEGF levels at day 25 measured in excess of 40 pg/ml. The increased levels observed on day 25 may be a consequence of loss of vascular integrity in the peritoneal blood vessels. It is known that VEGF can increase vascular permeability in these blood vessels, a process that is associated C D with increased influx of macromolecules in the plasma compartment into the peritoneal 32,33 cavity. Expression of VEGF in tumors derived from s.c. injection of MDA-MB-435 and MDA4345/LCC6 cells are shown in Figure 2. Sections derived from A431 tumors were used as a positive control for VEGF expression (Fig. 2A). VEGF protein staining of LCC6 tumor (Fig. 2E) was less than that observed E F for A431, but substantially greater than that observed for tumors derived from the parental MDA-MB-435 cells (Fig. 2C). It should be noted that plasma VEGF was not detected in animals bearing solid tumors derived from the LCC6 cell line. HER-2/neu expression was determined by western analysis and flow cytometric analysis of parental and MDA435/LCC6 where the highly overexpressing SK-BR-3 was used as a positive control (results not shown). These data suggested that there was a minor 1.3 fold increase in the level of HER-2/neu protein in the LCC6, as com- pared to parental. However, MDA-MB-435 Figure 3. Plasma antisense, lipid and antisense to lipid ratios following i.v. or i.p. injection of free or lipo- HER-2/neu expression was only 0.7% that somal H2A-ASO at an antisense dose of 10 mg/kg into tumor-free female SCID/Rag2m mice. (A, B) free of SKBR-3 cells (a cell line that overexpresses and liposomal antisense plasma levels following i.v. or i.p. injection, respectively. (C, D) plasma lipid levels HER-2/neu due to gene amplification), and following i.v. or i.p. injections, respectively. (E, F) plasma antisense to lipid ratios following i.v. or i.p. injec- LCC6 HER-2/neu expression was only 1% tions, respectively, of liposomal antisense. (∇) liposomal ASO; (") free ASO. Results are the mean of 4 ± that of SK-BR-3 (results not shown). These standard error of the mean. cell lines may be considered as low expressors of p185 (HER-2/neu protein). As seen in RESULTS Figure 2, panels D (MDA-MB-435) and F (MDA435/LCC6), tumors derived from these cell lines did not express sufficient levels of p185 for Assessment of VEGF and HER-2/neu Status in the MDA435/LCC6 detection by immunohistochemistry. The HER-2/neu positive SK-BR-3 Tumor Model. The MDA435/LCC6 tumor model is a more rapidly growing tumors are shown in panel B for comparison. tumor than the parental MDA-MB-435, with doubling times of 12 and 15.5 Plasma Elimination and Tissue Distribution of i.v. or i.p. Injected Free days, respectively, when grown as solid tumors (data not shown). In addition, or Liposomal Antisense. The data provided thus far suggest that the the MDA435/LCC6 can grow as an ascitic tumor and was isolated from a MDA435/LCC6 model could be used to measure the effects of agents tar- metastasis of a MDA-MB-435 cell derived tumor. Initial assessment of geting VEGF secretion. In addition, we have reported that ASOs targeting VEGF secretion was in supernatants collected from confluent cells grown in HER-2/neu may have therapeutic effects provided that the expression levels tissue culture. This data, shown in Figure 1A, indicates that there was a are low. With this in mind, studies assessing the plasma elimination of free significant 2.9-fold increase in VEGF levels in the supernatant derived and liposomal ASOs targeting VEGF and HER-2/neu were completed. from MDA435/LCC6 cells when compared to levels produced by the The plasma elimination of antisense, free or liposomal, was determined parental line. To determine if this increased VEGF level was also seen in after i.v. and i.p. administration and the results summarized in Figure 3. vivo, ELISAs on serum and ascites fluid of mice bearing ascitic Plasma antisense levels were measured using tracer levels of [ H]-H2A ASO MDA435/LCC6 tumors (Fig. 1B) were performed, and tumor sections and plasma liposomal lipid levels were determined through the use of [ C] taken from mice bearing solid tumors developed following s.c. injection of labeled CHE. Therefore, it is important to note that the antisense levels MDA-MB-435 and MDA4345/LCC6 cells were immunohistochemically measured may not represent intact sequence. The plasma antisense levels stained for VEGF expression (Fig. 2). The VEGF levels in the ascites fluid obtained following i.v. (Fig. 3A) and i.p. (Fig. 3B) injection of free (open collected from mice bearing ascitic MDA435/LCC6 tumors (Fig. 1B) circles) or liposome encapsulated (open triangles) H2A-ASO following a increased as the tumor progressed. Between day 21 and day 28 there was single i.v. antisense dose of 10 mg/kg, clearly illustrate the primary benefits 200 Cancer Biology & Therapy 2004; Vol. 3 Issue 2 © Landes Bioscience 2004. Not for distribution. ANTISENSE PK IN THE MDA435/LCC6 BREAST CANCER MODEL Table 1 TISSUE ANTISENSE LEVELS IN MDA435/LCC6 ASCITES TUMOR-BEARING MICE Time Post Injection Tissue 1 hour 4 hours 24 hours Liver 14.29 ± 1.27 18.72 ± 0.79 19.38 ± 0.93 Lung 3.18 ± 0.54 2.36 ± 0.10 1.10 ± 0.07 Spleen 13.37 ± 1.79 14.43 ± 1.35 12.07 ± 1.12 Kidney 50.35 ± 9.11 61.58 ± 1.64 64.05 ± 5.98 Muscle 1.02 ± 0.33 0.94 ± 0 0.84 ± 0.1 Antisense was administered on day 17 post tumor cell inoculation, with results with expressed as µg ASO per g tissue, following I.V. injection of 10 mg/kg VEGF-ASO (Mean of 4 ± standard error of the mean). mean AUC (1219.7 µg h/ml) were observed when the liposomal formu- B 0-24h lation was given by i.p. injection (Fig. 3B). As expected, the levels in the plasma at the 1 h time point following i.p injection were significantly (p < 0.005) lower than those obtained following i.v. administration. At 4 h, the level of ASO in the plasma was equivalent regardless of whether the route of administration was i.p. or i.v. and this is consistent with the observations that liposomes given i.p. rapidly enter the blood via the lymphatics (Bally MB; unpublished observation). The plasma liposomal lipid levels (Fig. 3C and D) indicate that the lipo- somal formulation, when given i.v., exhibits extended circulation lifetimes. As much as 20% of the injected lipid dose is still present in the plasma compartment 24 h after injection. The plasma levels of lipid obtained after i.p. injection are lower and less than 10% of the injected lipid dose can be found in the plasma compartment at 24 h. Perhaps the most important parameter that can be derived for a liposomal delivery system arises when calculating changes in entrapped content to liposomal lipid ratio over time following parenteral administration. This Figure 4. Serum antisense levels in MDA435/LCC6 ascites tumor-bearing parameter can be used to assess whether the therapeutically active agent dis- female SCID/Rag2m mice following injection of free or liposomal (A) sociates from the carrier system after administration. Figure 3E and F H2A-ASO or (B) VEGF-ASO. Antisense was administered on day 17 post includes this analysis, where the ratio of measured plasma ASO concentration tumor cell inoculation. (∇) liposomal ASO; (") free ASO. Results are the and measured plasma liposomal lipid concentration is plotted as a function of mean of 4 ± standard error of the mean. time after injection. A decrease in the ASO to lipid ratio as shown in Figure 3E and F indicates antisense release (dissociation) from the liposomes. attributable to use of an appropriately designed liposome delivery system. Approximately 20% of the associated ASOs are released from the carrier over Free antisense oligonucleotides were eliminated rapidly following i.v. injec- the 24 hour time period. It is assumed that ASO released from the liposomes tion, with greater than 95% of the injected dose eliminated within one is rapidly eliminated therefore the ASO measured is primarily due to the lipo- hour. When free ASO was given by i.p. injection, the blood levels at one somally encapsulated drug. hour were slightly higher than that achieved following i.v. administration Plasma elimination studies were also conducted in MDA435/LCC6 and this is reflected by the slight increase in mean AUC (153.8 µg h/ml tumor bearing SCID mice, where mice were injected i.p. with 2 x 10 0-24h following i.p. injection and 148 µg h/ml following i.v. injection). In contrast, MDA435/LCC6 cells and 17 days later they were injected i.v. with either when the liposomal formulation was given i.v., there were significant increases free or liposomal antisense at an antisense dose of 10 mg/kg. The plasma in circulating blood levels of ASOs at every time point evaluated, resulting ASO levels have been summarized in Figure 4 for both the HER-2/neu in mean plasma AUC of 1565.7 µg h/ml. Similar increases in plasma targeted (Fig. 4A) and the VEGF targeted (Fig. 4B) ASO sequences evaluated. 0-24h Table 2 TISSUE ANTISENSE AND LIPID LEVELS IN MDA435/LCC6 ASCITES TUMOR-BEARING MICE Antisense Lipid Time (hrs) 1 4 24 1 4 24 Liver 41.4 ± 1.3 48.8 ± 4.3 54.3 ± 11 557.9 ± 36.5 610.8 ± 115.7 805.1±240.2 Lung N.D.* 2.4 ± 1.3 2.3 ± 0.6 N.D. 30.3 ± 41.0 31.6±24.7 Spleen 92.2 ± 9.4 148.5 ± 17.2 138.7 ± 29.2 884.7 ± 182.9 755.9 ± 184.3 1454±225.9 Kidney 5.5 ± 1.0 9.3 ± 0.6 14.0 ± 1.9 N.D. N.D. 36.24±13.5 Muscle N.D. N.D. N.D. N.D. N.D. N.D. Antisense ws administered day 17 post tumor cell inoculation, with results expressed as µg per g tissue, following I.V. injection of liposomal VEGF-ASO at an antisense dose of 10 mg/kg (Mean ± standard error of the mean). *N.D. Below detectable limits which were set a a dpm value 2 times greater than background. www.landesbioscience.com Cancer Biology & Therapy 201 © Landes Bioscience 2004. Not for distribution. ANTISENSE PK IN THE MDA435/LCC6 BREAST CANCER MODEL liposomal ASO and spleen levels are 7 to 10-fold greater, depending on the time point. The increase in spleen and liver delivery is due in part to the natural tendency of liposomes to accumulate in these organs, even when prepared with surface grafted PEGs. Second, the tissue level of ASO in the kidney is at least 5-fold lower when the ASO is given in the liposomal form, an observation that is consistent with the fact that liposomes do not readily accumulate in organs such as the kidney (liposomal lipid levels were below detectable limits at the 1 and 4 hour time points) (data not shown). Similarly, the muscle ASO and lipid levels are below detectable limits, a consequence of little or no liposome accumulation in muscle tissue. It can be concluded from the data in Table 2 that the distribution of ASO, when given in the liposomal formulation is dictated by the biodistribution behav- ior of the liposomes. It is worth noting that the ASO to lipid ratios, estimated on the basis of the biodistribution data in Table 2, are lower than those measured in the plasma compartment (see Fig. 3E) and suggest that the ASO is lost from the liposomes both during and after tissue localization. Interestingly, the ASO to lipid ratio in the kidney, (estimated at the 24 h time point to be 0.4), is almost 3-fold higher that that measured in the plasma compartment. This is consistent with the fact that the kidney is the primary organ for free ASO removal, even after the ASO has been released from the liposomes. One of the benefits of utilizing a tumor model where the cells grow intraperitoneally within ascites fluid is that it provides easy access to the tumor and host cell populations residing within the peritoneal cavity and this, in turn, facilitates analysis of drug delivery and of associated therapeutics effects. To assess antisense delivery to the peritoneal cavity, MDA435/LCC6 tumor bearing animals (prepared as described for the studies shown in Fig. 4) were given an i.v. injection of free or liposomal VEGF-ASO on day 17 post-tumor cell inoculation. At 1, 4 and 24 hours post antisense injection, peritoneal fluid and associated cells were recovered by peritoneal lavage, as described in the Methods. There are two important conclusions that can be made on the basis of the data in Figure 5. First, the levels of ASO in the cell-free peritoneal fluid are comparable regardless of whether ASO has Figure 5. Peritoneal fluid (A) and peritoneal cell-associated (B) antisense lev- been given as a free or liposomal formulation. This result was surprising els following administration of free or liposomal VEGF-ASO at an antisense considering that there is a substantial increase in the blood levels and circu- dose of 10 mg/kg into female SCID/Rag2m MDA435/LCC6 ascites lation lifetime for the liposomal formulation, but are consistent with the tumor-bearing mice. Antisense was administered day 17 post tumor cell observations of others that the use of surface grafted PEGs can actually inoculation. (∇) liposomal ASO; (") free ASO. Results are the mean of 4 ± standard error of the mean. decrease extravasation rates and extent. For conventional small molecule anticancer drugs delivery in a PEG-free liposomal carrier typically results The data in Figure 4A is comparable to the plasma elimination data obtained in substantial increases in peritoneal cavity delivery, both in ascites tumor bearing and tumor free animals. It should be noted that the level of delivery in tumor free mice, suggesting that the presence of an established tumor did not impact the elimination of either the free or liposomal ASO. In addition, observed in the MDA435/LCC6 model is quite low, with as little as 1% of the injected dose accessing the peritoneal cavity at 24 h. The second impor- the data in Figure 4 suggests that the plasma elimination behavior of both ASO sequences are comparable, even though the HER-2/neu targeted ASO tant conclusion made regarding the data in Figure 5 concerns the increase in peritoneal cell delivery achieved when using the liposomal formulation, a is a 15 nucleotide sequence and the VEGF targeted ASO is a 21 nucleotide sequence. As was the case for non-tumor-bearing mice, the encapsulated result that is surprising considering that the presence of surface grafted PEGs should interfere with cell delivery. Although the error in this analysis antisense exhibited a much greater plasma residence time, with 4 hour levels being approximately 50-fold higher for liposomal formulation as compared is substantial and significant differences are only notable at the 4 h time point (p < 0.05), it is evident that there is an apparent increase in cell delivery. to free. Mean AUC0-24 analysis of these data demonstrate that the liposomal formulation provides for a 10-fold increase in mean plasma AUC0-24 when As much as 3 to 5 times greater levels of ASO are associated with the isolated peritoneal cells following administration of the liposomal formulation. compared to the free ASO. The tissue distribution of ASO and liposomal lipid following i.v. admin- Approximately 2 x 10 cells are isolated from the peritoneal cavity under the conditions employed in these studies (Fig. 6A) and this is a 30-fold increase istration of the VEGF-ASO was also determined and the results have been summarized in Tables 1 (free ASO) and 2 (liposomal ASO). There are two in cellularity when compared to nontumor bearing mice (data not shown). This cell number dropped to 1.4 x 10 when mice were treated with unen- important conclusions that can be drawn on the basis of the data in Table 1. First, the highest tissue levels of ASO obtained after administration of the capsulated ASO targeted to HER-2/neu, and further to levels comparable to nontumor bearing mice following treatment with liposomally formulated unencapsulated form was in the kidney. This is consistent with previous studies [20] and is indicative of rapid renal clearance of this agent. Second, HER2-ASO (Fig. 6A). Under conditions where the tumor bearing animals have been treated with saline (control treatment groups), almost 40% of the the levels of ASO in the spleen and liver are equivalent on a weight basis and the levels within these tissues are reasonably constant over the 24 h time cells isolated from the peritoneal cavity are murine (i.e., nonhuman) as judged by flow cytometric analysis. The influx of murine cells, primarily course. Liver accumulation of the injected ASO accounts for less than 10% of the injected ASO dose, while the spleen accumulation accounts for less neutrophils, into the peritoneal cavity is probably a consequence of tumor growth and perhaps the associated production of VEGF. As shown in Figure than 1% of the injected dose. When comparing these data to the liposomal formulation (Table 2), there are several notable differences. First, liver and 6B, of the total number of cells contained within the peritoneal cavity of saline treated mice, only 66% are viable, and of those, approximately 60% spleen ASO levels are substantially higher when the ASO is given in lipo- somal formulation. Liver levels are almost 3-fold greater in animals given were of human origin. Following treatment with free HER2-ASO, the 202 Cancer Biology & Therapy 2004; Vol. 3 Issue 2 © Landes Bioscience 2004. Not for distribution. ANTISENSE PK IN THE MDA435/LCC6 BREAST CANCER MODEL VEGF and HER-2/neu are just two A B examples of the many new molecular targets being researched for novel anti- cancer therapeutics. The agents targeting these molecules are also novel, with agents such as recombinant DNA, ASOs and monoclonal antibodies becoming predominant. These new agents can often not be administered systemically without the use of a cosol- vent or carrier systems of some sort, such as polymers, lipid complexes or liposomes. As is true for drugs such as anti-fun- 37 38 gals, anti-bacterials and several anti- 39-42 cancer agents, various ASO are being developed as liposomal formulations in order to protect the therapeutic agent 43-45 from degradation. A commonly used modification of ASO is the chemical variation of the backbone, the most commonly used being that of the substi- tution of a sulfur atom for one of the nonbridging oxygen atoms in the phos- Figure 6. Tumor burden (A) and percent murine (green)/human (red) cells (B) in MDA435/LCC6 tumor bearing phodiester bond. This phosphorothioate female SCID/Rag2m mice following treatment with free H2A-ASO or liposomal H2A-ASO. Results are the bond increases the serum terminal mean ± standard error of the mean of a minimum of 4 animals. half-life of ASO from minutes, to up to 9 percentage of viable cells in the total count did not change significantly, nor hours, a result that is primarily a consequence of improved stability did the ratio of murine to human cells, however a striking reduction in the against nucleases within the circulation. Liposomes are advantageous number of viable human cells was noted following treatment with liposo- for the delivery of many therapeutics as they are able to extend the mally encapsulated HER2-ASO, with less than 5% of the total cell count circulation lifetime as well as engendering significant increases in the being made up of viable human cells, with the remaining being composed plasma concentration of the associated agent. The increased circula- of murine cells (Fig. 6B). This result demonstrated not only a reduction in tion lifetime and plasma concentration have been shown to effect the total number of cells, but also a specific reduction in the number of increases in drug accumulation at sites of disease, particularly to human tumor cells within the peritoneal cavities of these mice. those diseases which cause damage to the surrounding blood vessels. This is particularly true for sites of tumor growth where there is DISCUSSION induction of neovasculature and the release of factors, such as VEGF, which cause increases in blood vessel permeability to circulating An assessment of the potential clinical suitability of novel anticancer 31-33 macromolecules. Studies demonstrating the therapeutic activity therapeutics typically includes the determination of anti-tumor of ASOs in vivo have had to rely on prolonged drug administration, activity in preclinical animal models of cancer, most of which are therefore it is anticipated that liposomal formulations of ASO mouse based. Ideally, the anti-tumor response to the agent being should improve therapy by increasing delivery of the intact sequence tested should assist in predicting activity when used in a clinical setting. to sites of disease and, perhaps, by reducing the frequency of drug An associated correlate to this ideal is that the model used should administration. also mimic the human disease state or condition. The MDA435/ The pharmacokinetic benefits of administering ASOs in a lipo- LCC6 ascites tumor model is a direct derivative of the estrogen somal formulation have been illustrated well by the data presented receptor negative, invasive and metastatic MDA-MB-435 cell in this report. Where the mean plasma AUC is more then 34-35 0-24h line and has been characterized as such by isotype and karyotype 10-fold greater than that achieved following i.v. administration of analysis. It has been suggested that the coexpression of molecular free ASO and the biodistribution of the ASO is dictated by the markers in neoplastic transformation functions as a significant prog- biodistribution of the liposomal carrier. It is important to note that nostic correlate for recurrence, and that the number of coexpressed the method to prepare the liposomal formulation of ASO used here markers is directly related to this significance. Given these points, has been reported elsewhere and these formulations are currently we chose to characterize the protein expression of VEGF and in preclinical development in our laboratory and others. HER-2/neu. Many tumor cells express and secrete VEGF at high levels, with References 1. Gasparini G, Toi M, Gion M, Verderio P, Dittadi R, Hanatani M, et al. Prognostic signif- the effect of inducing the formation of new vasculature to promote icance of vascular endothelial growth factor protein in node-negative breast carcinoma. J tumor growth. 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Petit AM, Rak J, Hung MC, Rockwell P, Goldstein N, Fendly B, et al. Neutralizing anti- bodies against epidermal growth factor and ErbB-2/neu receptor tyrosine kinases 37. Ahmad I, Perkins WR, Lupan DM, Selsted ME, Janoff AS. Liposomal entrapment of the down-regulate vascular endothelial growth factor production by tumor cells in vitro and in neutrophil-derived peptide indolicidin endows it with in vivo antifungal activity. Biochim vivo: Angiogenic implications for signal transduction therapy of solid tumors. Am J Pathol Biophys Acta 1995; 1237:109-14. 1997; 151:1523-30. 38. Webb MS, Boman NL, Wiseman DJ, Saxon D, Sutton K, Wong KF, et al. Antibacterial 10. Kerbel RS, Viloria-Petit A, Klement G, Rak J. ‘Accidental’ anti-angiogenic drugs. efficacy against an in vivo Salmonella typhimurium infection model and pharmacokinetics anti-oncogene directed signal transduction inhibitors and conventional chemotherapeutic of a liposomal ciprofloxacin formulation. Antimicrob Agents Chemother 1998; 42:45-52. agents as examples. Eur J Cancer 2000; 36:1248-57. 39. Gabizon A, Catane R, Uziely B, Kaufman B, Safra T, Cohen R, et al. Prolonged circulation 11. Yen L, You XL, Al Moustafa AE, Batist G, Hynes NE, Mader S, et al. Heregulin selective- time and enhanced accumulation in malignant exudates of doxorubicin encapsulated in ly upregulates vascular endothelial growth factor secretion in cancer cells and stimulates polyethylene-glycol coated liposomes. Cancer Res 1994; 54:987-92. angiogenesis. Oncogene 2000; 19:3460-9. 40. Genne P, Olsson NO, Gutierrez G, Duchamp O, Chauffert B. Liposomal mitoxantrone for 12. Pegram MD, Reese DM. Combined biological therapy of breast cancer using monoclonal the local treatment of peritoneal carcinomatosis induced by colon cancer cells in mice. antibodies directed against HER2/neu protein and vascular endothelial growth factor. Anticancer Drug Des 1994; 9:73-84. Semin Oncol 2002; 29(3 Suppl 11):29-37. 41. 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Pharmacodynamic Behavior of Liposomal Antisense Oligonucleotides Targeting Her-2/neu and Vascular Endothelial Growth Factor in an Ascitic MDA435/LCC6 Human Breast Cancer Model

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Taylor & Francis
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Copyright © 2003 Landes Bioscience
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1555-8576
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1538-4047
DOI
10.4161/cbt.3.2.622
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Abstract

[Cancer Biology & Therapy 3:2, 197-204, February 2004]; ©2004 Landes Bioscience Research Papers Pharmacodynamic Behavior of Liposomal Antisense Oligonucleotides Targeting Her-2/neu and Vascular Endothelial Growth Factor in an Ascitic MDA435/LCC6 Human Breast Cancer Model 1, ABSTRACT Dawn N. Waterhouse * Wieslawa H. Dragowska The nature of anti-cancer therapeutics is currently undergoing a paradigm change, with biologic agents slowly being introduced into the therapeutic armory, displacing or Karen A. Gelmon complimenting the traditionally used cytotoxic agents. These new agents include mono- 1,2 Lawrence D. Mayer clonal antibodies, recombinant DNA, antisense oligonucleotides (ASO) and others. To 1,3 Marcel B. Bally assess the new therapeutics, new predictive models are required. Utilizing the MDA435/LCC6 human breast cancer xenograft model, the pharmacokinetic behavior of British Columbia Cancer Research Centre; Department of Advanced Therapeutics; antisense oligonucleotides targeted against vascular endothelial growth factor and Vancouver, B.C., Canada HER-2/neu was assessed. For pharmacodynamic analysis, ASO in buffer or encapsulated University of British Columbia; Faculty of Pharmaceutical Sciences; Vancouver, B.C., in a liposomal formulation were injected intravenously or intraperitoneally into Canada MDA435/LCC6 ascites tumor-bearing mice. Plasma antisense elimination, tissue distri- University of British Columbia; Department of Pathology and Laboratory Medicine; bution, total peritoneal antisense and peritoneal cell associated antisense levels were Vancouver, B.C., Canada determined. Liposomal encapsulation led to significant decreases in the plasma elimination *Correspondence to: Dawn N. Waterhouse; British Columbia Cancer Research rate, as evidenced by an approximate 10-fold increase in mean AUC over 24 hours, as Centre; Department of Advanced Therapeutics; 601 West 10th Avenue; Vancouver, well as enhanced peritoneal cell delivery in mice bearing ascites tumors. Tissue distribution B.C., Canada; Tel.: 604.877.6011; Fax: 604.877.6011; Email: dwater@bccancer.bc.ca studies of both free and liposome encapsulated ASO indicated that ASO distribution was Received 09/16/03; Accepted 09/16/03 dictated primarily by the liposomal carrier when administered in liposomal form. Previously published online as a Cancer Biology & Therapy E-publication: http://www.landesbioscience.com/journals/cbt/abstract.php?id=622 INTRODUCTION KEY WORDS In North America and Europe, approximately 11% of all women, and 1% of all men pharmacokinetic, pharmacodynamic, antisense will develop breast cancer. Many of these patients will also develop metastatic disease, with oligonucleotide, liposome, xenograft tumor approximately 35% of those who develop breast cancer ultimately succumbing to their disease. For this reason, there is an obvious need for developing more efficacious treatment ABBREVIATIONS strategies. Research has elucidated several specific prognostic and predictive factors to ASO antisense oligonucleotides identify patients at high risk for more aggressive disease, metastasis and for recurrence of AUCarea under the curve disease in order to combat these statistics. Several studies have demonstrated, for example, CHE cholesteryl hexadecyl ether that an increase in cytosolic levels of vascular endothelial growth factor (VEGF) in tumor Chol cholesterol tissue samples is indicative of poorer prognosis for patients with node-negative breast DEAE diethylaminoethyl 1,2 3 carcinoma. VEGF plays a key role in the process of angiogenesis, and the extent of DMEM Dulbecco’s minimal essential 4-8 tumor angiogenesis is now widely accepted as a prognostic factor in several cancer types, medium DODAP dioleoyl dimethylammonium with higher levels being indicative of more aggressive disease. propane We believe that anti-VEGF treatment modalities will be of great interest in the context FBS fetal bovine serum of the drug combination treatment strategies being developed for treatment of molecular- H2A-ASO anti-HER-2/neu antisense ly defined aggressive disease. Investigators have demonstrated a link between VEGF and HBS HEPES Buffered saline HER-2/neu, another marker of poor prognosis in both node positive and node negative HBSS Hank’s balanced salt solution 9-14 breast cancers. Over-expression of HER-2/neu is found in 20–25% of breast cancer modified 15-18 patients, and is predictive of shorter disease free and overall survival. Research is also PEG polyethylene glycol elucidating a benefit from targeting both VEGF and HER-2/neu in conjunction in the PEG-C14CER PEG2000C14ceramide therapeutic intervention of breast cancer. VEGF vascular endothelial growth The discovery of new prognostic and predictive markers for breast cancer is obviously factor VEGF-ASO anti-VEGF antisense of great potential therapeutic value, however in order to exploit these markers in the devel- opment of treatment strategies, researchers require appropriate in vivo models. Thus, when ACKNOWLEDGEMENTS considering strategies targeting HER-2/neu and VEGF, the cell lines used to establish tumor models must also express the targets. The development of a human xenograft ascites This work was funded by a National Cancer model fulfilling some of these requirements has been achieved in the MDA435/LCC6 Institute of Canada, Breast Cancer Initiative grant. model. This model has a reproducible growth curve in immune compromised mice and D.N.W. is a Canadian Breast Cancer Foundation exhibits sensitivity to cytotoxic drugs known to be active in the treatment of human breast Research Fellow. cancer patients. The cells are easily maintained in vitro, thus allowing rapid assessment of drugs both in vitro and in vivo. In this report we provide a further characterization of the MDA435/LCC6 model for VEGF and HER-2/neu expression. www.landesbioscience.com Cancer Biology & Therapy 197 © Landes Bioscience 2004. Not for distribution. ANTISENSE PK IN THE MDA435/LCC6 BREAST CANCER MODEL The use of this model to assess the pharmacodynamic behavior of for antisense and 130 dpm/µg for lipid. Antisense and lipid solutions were briefly heated to 60˚C, then combined in a weight-to-weight ratio of 0.2 to two antisense oligonucleotide sequences designed to inhibit expres- 1.0, antisense being added drop-wise to lipids in ethanol while mixing sion of VEGF and HER-2/neu has been evaluated. ASOs have been vigorously. Mixtures were subjected to 5 freeze-thaw cycles, consisting of extensively researched as pharmaceutical agents, however even with immersion in liquid nitrogen followed by thawing at 65 C, prior to repeated the chemical modification of the phosphate backbone to a more (10X) extrusion through three stacked 100 nm pore size polycarbonate stable phosphorothioate bond, these ASO are rapidly eliminated membranes (Nucleopore, Pleasanton, CA) using a thermobarrel equipped from the circulation, primarily by the kidneys. Although ASOs are extrusion device (Northern Lipids Inc., Vancouver, BC). Following extrusion, 22-23 efficacious when given in vivo, activity is often dependent on samples were dialyzed against 300 mM citrate buffer, pH 4.0 for two hours use of aggressive dose scheduling involving daily injections for time to remove ethanol, then against pH 7.5 HEPES buffered saline (HBS) for a periods in excess of two weeks or use of micro-infusion pumps. minimum of 12 hours at 4 C. Liposomes were then passed over a diethy- Thus it may be argued that drug carrier formulations may engender laminoethyl (DEAE)-agarose anion exchange column (BioRad, Mississauga, Ontario, Canada) equilibrated in HBS to remove unencapsulated antisense. improved pharmacological properties including enhanced circula- The size of the resultant liposomes was typically about 130 nm as deter- tion longevity, as well as increased delivery of the ASO to sites where mined by analysis with a Nicomp 270 submicron particle sizer (Pacific the target cells are localized. The two antisense oligonucleotides Scientific, Santa Barbara, CA), operating at 632.8 nm. utilized in these studies were a 21-mer with a phosphorothioate As necessary, lipid and antisense concentrations were determined by backbone, targeted against human VEGF with demonstrated activity 14 3 [ C] and [ H] levels (respectively), by mixing samples with 5 mL of against production of this protein in an in vitro model, and a PicoFluor 40 scintillation cocktail (Packard, Groningen, The Netherlands), 15-mer phosphorothioate ASO specific for the area of HER-2/neu and counting with a Packard 1900 scintillation counter (Meriden, CT). 26-27 mRNA immediately upstream of the initiation codon. These Encapsulation of antisense was determined using scintillation counts or antisense sequences were encapsulated in a novel liposomal formula- by solubilization method as follows. A 10 µl aliquot of liposomal antisense tion, recently described by Semple et al. and the pharmacodynamic or water (for blank) was added to 240 µl distilled water. This was combined behavior of these ASOs, delivered in free or liposomal form, was with 750 µl CHCl /MeOH (1.0:2.1 vol:vol) and 100 µl MeOH. The mix- determined using the MDA435/LCC6 ascites model. tures were vortexed until clear, and absorbance determined at 260 nm. Concentration was determined according to the following calculation: Concentration = (A )(Extinction coefficient of antisense, ε)(dilution) MATERIALS AND METHODS Where ε = 34.5 µg/OD unit. In Vivo Studies. Female SCID/Rag2m mice (Taconic, Germantown, SK-BR-3, MCF7 and A431 cells were obtained from the American Type NY), n = 8, were injected i.p. with 2 x 10 MDA435/LCC6 cells, or s.c. Culture Collection (Rockville, MD). MDA-MB-435 cells were from the with 5 x 10 MDA435/LCC6 cells, in a total volume of 200 or 50 µL, tumor repository of the National Cancer Institute (Frederick, MD). respectively. These cell numbers give consistent and reproducible tumor MDA435/LCC6 cells were a generous gift from Dr. Robert Clarke, growth rates. Total volume of ascites fluid, ascites cell number, and ascites Georgetown University (Washington, DC). SK-BR-3 cells were maintained and serum VEGF levels were determined for the ascites model after 0, 7, 14, in McCoy’s 5A medium with 10% fetal bovine serum (FBS), MCF7 were 21 or 28 days. Immunohistochemical analysis was performed on sections maintained in RPMI 1640, 10% FBS and 2 mM L-glutamine. All other derived from solid tumors and these sections were stained for VEGF, cells were maintained in Dulbecco’s minimal essential medium (DMEM) HER-2/neu and bcl-2 proteins as described below. For determination of supplemented with 10% FBS and 2 mM L-glutamine. Cultures were sus- tained at 37 C and in an atmosphere of 5% CO . All media, Hank’s ascites VEGF levels, an enzyme linked immunosorbent assay (ELISA) (R&D Systems) was used, using standards for serum VEGF levels. To assess Balanced Salt Solution Modified (HBSS), and trypsin-EDTA were obtained from StemCell Technologies Inc. (Vancouver, BC, Canada). levels of VEGF in cells grown in culture, cells were plated in 25 cm tissue culture flasks. When cells were confluent, supernatant was collected and Phosphorothioate backbone anti-HER-2/neu antisense oligonucleotide (H2A-ASO) and anti-VEGF-A antisense oligonucleotide (VEGF-ASO) assayed by VEGF ELISA (Quantikine human VEGF quantitative colori- metric sandwich ELISA, R&D Systems, Minneapolis, MN). were synthesized by Hybridon Inc. (Cambridge, MA). Sequences of anti- sense oligonucleotides were, H2A-ASO: 5’-GGT GCT CAC TGC GGC Ascites fluid was collected by peritoneal lavage. For early time-points, a known volume of HBSS was injected into the peritoneum prior to lavage. and VEGF-ASO: 5’- AGA CAG CAG AAA GTT CAT GGT-3’, targeting the 15 bases immediately upstream of the initiation codon of human Cells were counted with a hemacytometer. For the s.c. model, tumor size was determined by the formula L(W )/2, with measurements being taken in HER-2/neu mRNA, and -3 to +18 of the human vascular endothelial 3 3 growth factor mRNA coding region, respectively. [ H]-VEGF-ASO and mm with digital calipers. This yielded tumor volume in mm , which we have determined to be roughly equivalent to tumor weight in grams. When [ H]-H2A-ASO were obtained from TriLink Biotechnologies, Inc. (San Diego, CA). [14C]-cholesteryl hexadecyl ether (CHE) was obtained from tumors reached a size of between 0.5 and 1 g, the animals were terminated, and tumors collected in formalin. Tumors were embedded in paraffin, and Amersham Canada Ltd. (Oakville, ON, Canada). All chemicals or reagents not otherwise mentioned were from Sigma (Oakville, ON, Canada). 5 µm sections prepared (Wax-It, Aldergrove, BC). Sections were stained for Preparation of Liposomal Antisense Oligonucleotides. Liposomal anti- VEGF using mouse anti-human VEGF monoclonal antibody, clone sense was prepared following the method of Semple et al. Stock lipid solu- 26503.11 from R&D Systems. Blocking agent and secondary antibody were tions were prepared in 100% ethanol. 1,2-distearoyl-sn-glycero-3-phospho- from the Vector Laboratories Vectastain Elite mouse ABC kit (Burlingame, choline (DSPC) (Northern Lipids Inc., Vancouver, BC), cholesterol (Chol) CA). Visualization was with ImmunoPure Metal Enhanced DAB (Sigma) and dioleoyl dimethylammonium propane (DODAP) (Avanti Polar Substrate kit (Pierce, Rockford, IL). Positive controls for VEGF staining Lipids, Alabaster, AL) were prepared at 20 mg/ml, while PEG2000 were sections of tumors derived following s.c. injection of 2 x 10 A431 C14Ceramide (PEG-C CER)(Northern Lipids Inc.) was prepared at 50 cells. mg/ml. Lipids were combined in a molar ratio of 2.0:2.5:4.5:1.0 Immunohistochemical staining for HER-2/neu was performed using an 14 30 (DODAP:DSPC:Chol:PEG-C -CER). As required, [ C]-CHE was added antibody cocktail, as described by Paik et al. Primary antibodies were poly- to the dissolved lipids as a nonexchangeable, nonmetabolizable marker. clonal rabbit anti-c-erbB-2 and monoclonal mouse anti-HER2 (c-erbB-2) Antisense was dissolved in 300 mM citrate buffer, pH 4.0 at a concentration antibody (Zymed Laboratories). Secondary antibodies and blocking solu- 3 3 of 3.33 mg/ml, with the addition of sufficient [ H]-H2A-ASO or [ H]- tions were an equal volume mixture from the Vector Laboratories VEGF-ASO for quantitation. Typical specific activities were 900 dpm/µg Vectastain Elite mouse and rabbit ABC kits. Slides were counterstained 198 Cancer Biology & Therapy 2004; Vol. 3 Issue 2 © Landes Bioscience 2004. Not for distribution. ANTISENSE PK IN THE MDA435/LCC6 BREAST CANCER MODEL Figure 1. (A) In vitro VEGF levels in supernatant collected from confluent MDA-MB-435 and MDA435/LCC6 cells following 24 hours incubation of matched cell number, measured by ELISA. Results in pg/ml ± standard error of the mean, and (B) in vivo VEGF levels from peritoneal fluid of MDA435/LCC6 derived ascites tumors (female SCID/Rag2m mice), collect- Figure 2. Immunohistochemical assessment of in vivo VEGF and HER-2/neu ed 0, 7, 14, 21 or 28 days post tumor cell inoculation. Insert shows serum levels. 5 x 10 A431 (A), SK-BR-3 (B), MDA-MB-435 (C, D) or VEGF concentration from these same animals. Ascites and serum were MDA435/LCC6 (E, F) were inoculated s.c. into female SCID/Rag2 mice. assayed by ELISA; results show pg and pg/ml, respectively, ± standard error Tumors were collected in formalin before reaching 1 gram in size, paraffin of the mean of 4 animals. embedded and stained immunohistochemically for VEGF (A, C and E) or HER-2/neu (B, D and F). Panels (A) and (B) are positive controls for VEGF and with Gill’s hematoxylin, dehydrated in ethanol, cleared in xylene and HER-2/neu staining, respectively. Visualization is with diaminobenzidine, mounted. Positive control sections for HER-2/neu staining were sections of where brown areas indicate positive staining. Tissues were counterstained tumors derived following s.c. injection of SK-BR-3 cells. with Gill’s hematoxylin. To determine the circulation longevity of the liposome encapsulated antisense in comparison to free antisense, groups of four female SCID/Rag2 counting. The liver, spleen, lung, kidney, heart and muscle were collected mice were injected i.v. (200 µl) or i.p. (500 µl) with either free or liposomal and weighed. Tissues (with the exception of the spleen) were made into a antisense at an antisense dose of 10 mg/kg. Samples were prepared with 30% homogenate in water using a Polytron tissue homogenizer. Two 3 3 14 [ H]-VEGF-ASO or [ H]-H2A-ASO and [ C]-CHE lipid label for quan- hundred microliters of this homogenate was transferred to scintillation vials. titation. Blood was collected by tail nick at 1 and 4 hours, and by cardiac Spleens were collected directly into scintillation vials. Solvable (500 µl) puncture at 24 hours. Serum antisense and liposomal lipid levels were was added to each vial, which were then incubated at 50°C overnight. The assessed by radiometric assay. This experiment was repeated in tumor bear- samples were cooled to room temperature prior to addition of 50 µL 200 ing mice. Female SCID/Rag2m mice were injected i.p. on day 0 with 2 x mM EDTA, 25 µl 10 M HCl and 200 µL 30% H O . This mixture was 2 2 10 MDA435/LCC6 cells. On day 17, free or liposomal antisense was incubated at room temperature for one hour prior to addition of 5 mL 3 3 14 injected. In these studies the mice were injected i.v. with [ H]-VEGF-ASO scintillation cocktail. Samples were analyzed for [ H] and [ C] by scintilla- 3 14 or [ H]-H2A-ASO and [ C]-CHE labeled liposomal antisense, at an anti- tion counting, and values corrected for tissue blood content. sense concentration of 10 mg/kg. At 1, 2, 4, 24 and 48 hours, groups of 4 Statistical analysis was performed with ANOVA one way post-hoc mice were sacrificed. Blood was collected by cardiac puncture. Peritoneal comparisons. lavages were performed by first injecting 3 ml HBSS into the peritoneal cavity and the abdomen was gently massaged for a period of 30 to 60 seconds. Subsequently the peritoneal fluid, with associated cells was carefully collected into sterile polystyrene tubes. Total volume was estimated and cell number determined using a hemocytometer. Aliquots (1 ml) were taken for scintillation www.landesbioscience.com Cancer Biology & Therapy 199 © Landes Bioscience 2004. Not for distribution. ANTISENSE PK IN THE MDA435/LCC6 BREAST CANCER MODEL a decline in the number of viable tumor cells in the peritoneum (data not shown) and this decrease in cell number correlated A B with the decline in peritoneal VEGF levels. As indicated in the insert to Figure 1B, VEGF levels in the serum of these mice steadily increased over the time period of tumor growth, with VEGF levels at day 25 measured in excess of 40 pg/ml. The increased levels observed on day 25 may be a consequence of loss of vascular integrity in the peritoneal blood vessels. It is known that VEGF can increase vascular permeability in these blood vessels, a process that is associated C D with increased influx of macromolecules in the plasma compartment into the peritoneal 32,33 cavity. Expression of VEGF in tumors derived from s.c. injection of MDA-MB-435 and MDA4345/LCC6 cells are shown in Figure 2. Sections derived from A431 tumors were used as a positive control for VEGF expression (Fig. 2A). VEGF protein staining of LCC6 tumor (Fig. 2E) was less than that observed E F for A431, but substantially greater than that observed for tumors derived from the parental MDA-MB-435 cells (Fig. 2C). It should be noted that plasma VEGF was not detected in animals bearing solid tumors derived from the LCC6 cell line. HER-2/neu expression was determined by western analysis and flow cytometric analysis of parental and MDA435/LCC6 where the highly overexpressing SK-BR-3 was used as a positive control (results not shown). These data suggested that there was a minor 1.3 fold increase in the level of HER-2/neu protein in the LCC6, as com- pared to parental. However, MDA-MB-435 Figure 3. Plasma antisense, lipid and antisense to lipid ratios following i.v. or i.p. injection of free or lipo- HER-2/neu expression was only 0.7% that somal H2A-ASO at an antisense dose of 10 mg/kg into tumor-free female SCID/Rag2m mice. (A, B) free of SKBR-3 cells (a cell line that overexpresses and liposomal antisense plasma levels following i.v. or i.p. injection, respectively. (C, D) plasma lipid levels HER-2/neu due to gene amplification), and following i.v. or i.p. injections, respectively. (E, F) plasma antisense to lipid ratios following i.v. or i.p. injec- LCC6 HER-2/neu expression was only 1% tions, respectively, of liposomal antisense. (∇) liposomal ASO; (") free ASO. Results are the mean of 4 ± that of SK-BR-3 (results not shown). These standard error of the mean. cell lines may be considered as low expressors of p185 (HER-2/neu protein). As seen in RESULTS Figure 2, panels D (MDA-MB-435) and F (MDA435/LCC6), tumors derived from these cell lines did not express sufficient levels of p185 for Assessment of VEGF and HER-2/neu Status in the MDA435/LCC6 detection by immunohistochemistry. The HER-2/neu positive SK-BR-3 Tumor Model. The MDA435/LCC6 tumor model is a more rapidly growing tumors are shown in panel B for comparison. tumor than the parental MDA-MB-435, with doubling times of 12 and 15.5 Plasma Elimination and Tissue Distribution of i.v. or i.p. Injected Free days, respectively, when grown as solid tumors (data not shown). In addition, or Liposomal Antisense. The data provided thus far suggest that the the MDA435/LCC6 can grow as an ascitic tumor and was isolated from a MDA435/LCC6 model could be used to measure the effects of agents tar- metastasis of a MDA-MB-435 cell derived tumor. Initial assessment of geting VEGF secretion. In addition, we have reported that ASOs targeting VEGF secretion was in supernatants collected from confluent cells grown in HER-2/neu may have therapeutic effects provided that the expression levels tissue culture. This data, shown in Figure 1A, indicates that there was a are low. With this in mind, studies assessing the plasma elimination of free significant 2.9-fold increase in VEGF levels in the supernatant derived and liposomal ASOs targeting VEGF and HER-2/neu were completed. from MDA435/LCC6 cells when compared to levels produced by the The plasma elimination of antisense, free or liposomal, was determined parental line. To determine if this increased VEGF level was also seen in after i.v. and i.p. administration and the results summarized in Figure 3. vivo, ELISAs on serum and ascites fluid of mice bearing ascitic Plasma antisense levels were measured using tracer levels of [ H]-H2A ASO MDA435/LCC6 tumors (Fig. 1B) were performed, and tumor sections and plasma liposomal lipid levels were determined through the use of [ C] taken from mice bearing solid tumors developed following s.c. injection of labeled CHE. Therefore, it is important to note that the antisense levels MDA-MB-435 and MDA4345/LCC6 cells were immunohistochemically measured may not represent intact sequence. The plasma antisense levels stained for VEGF expression (Fig. 2). The VEGF levels in the ascites fluid obtained following i.v. (Fig. 3A) and i.p. (Fig. 3B) injection of free (open collected from mice bearing ascitic MDA435/LCC6 tumors (Fig. 1B) circles) or liposome encapsulated (open triangles) H2A-ASO following a increased as the tumor progressed. Between day 21 and day 28 there was single i.v. antisense dose of 10 mg/kg, clearly illustrate the primary benefits 200 Cancer Biology & Therapy 2004; Vol. 3 Issue 2 © Landes Bioscience 2004. Not for distribution. ANTISENSE PK IN THE MDA435/LCC6 BREAST CANCER MODEL Table 1 TISSUE ANTISENSE LEVELS IN MDA435/LCC6 ASCITES TUMOR-BEARING MICE Time Post Injection Tissue 1 hour 4 hours 24 hours Liver 14.29 ± 1.27 18.72 ± 0.79 19.38 ± 0.93 Lung 3.18 ± 0.54 2.36 ± 0.10 1.10 ± 0.07 Spleen 13.37 ± 1.79 14.43 ± 1.35 12.07 ± 1.12 Kidney 50.35 ± 9.11 61.58 ± 1.64 64.05 ± 5.98 Muscle 1.02 ± 0.33 0.94 ± 0 0.84 ± 0.1 Antisense was administered on day 17 post tumor cell inoculation, with results with expressed as µg ASO per g tissue, following I.V. injection of 10 mg/kg VEGF-ASO (Mean of 4 ± standard error of the mean). mean AUC (1219.7 µg h/ml) were observed when the liposomal formu- B 0-24h lation was given by i.p. injection (Fig. 3B). As expected, the levels in the plasma at the 1 h time point following i.p injection were significantly (p < 0.005) lower than those obtained following i.v. administration. At 4 h, the level of ASO in the plasma was equivalent regardless of whether the route of administration was i.p. or i.v. and this is consistent with the observations that liposomes given i.p. rapidly enter the blood via the lymphatics (Bally MB; unpublished observation). The plasma liposomal lipid levels (Fig. 3C and D) indicate that the lipo- somal formulation, when given i.v., exhibits extended circulation lifetimes. As much as 20% of the injected lipid dose is still present in the plasma compartment 24 h after injection. The plasma levels of lipid obtained after i.p. injection are lower and less than 10% of the injected lipid dose can be found in the plasma compartment at 24 h. Perhaps the most important parameter that can be derived for a liposomal delivery system arises when calculating changes in entrapped content to liposomal lipid ratio over time following parenteral administration. This Figure 4. Serum antisense levels in MDA435/LCC6 ascites tumor-bearing parameter can be used to assess whether the therapeutically active agent dis- female SCID/Rag2m mice following injection of free or liposomal (A) sociates from the carrier system after administration. Figure 3E and F H2A-ASO or (B) VEGF-ASO. Antisense was administered on day 17 post includes this analysis, where the ratio of measured plasma ASO concentration tumor cell inoculation. (∇) liposomal ASO; (") free ASO. Results are the and measured plasma liposomal lipid concentration is plotted as a function of mean of 4 ± standard error of the mean. time after injection. A decrease in the ASO to lipid ratio as shown in Figure 3E and F indicates antisense release (dissociation) from the liposomes. attributable to use of an appropriately designed liposome delivery system. Approximately 20% of the associated ASOs are released from the carrier over Free antisense oligonucleotides were eliminated rapidly following i.v. injec- the 24 hour time period. It is assumed that ASO released from the liposomes tion, with greater than 95% of the injected dose eliminated within one is rapidly eliminated therefore the ASO measured is primarily due to the lipo- hour. When free ASO was given by i.p. injection, the blood levels at one somally encapsulated drug. hour were slightly higher than that achieved following i.v. administration Plasma elimination studies were also conducted in MDA435/LCC6 and this is reflected by the slight increase in mean AUC (153.8 µg h/ml tumor bearing SCID mice, where mice were injected i.p. with 2 x 10 0-24h following i.p. injection and 148 µg h/ml following i.v. injection). In contrast, MDA435/LCC6 cells and 17 days later they were injected i.v. with either when the liposomal formulation was given i.v., there were significant increases free or liposomal antisense at an antisense dose of 10 mg/kg. The plasma in circulating blood levels of ASOs at every time point evaluated, resulting ASO levels have been summarized in Figure 4 for both the HER-2/neu in mean plasma AUC of 1565.7 µg h/ml. Similar increases in plasma targeted (Fig. 4A) and the VEGF targeted (Fig. 4B) ASO sequences evaluated. 0-24h Table 2 TISSUE ANTISENSE AND LIPID LEVELS IN MDA435/LCC6 ASCITES TUMOR-BEARING MICE Antisense Lipid Time (hrs) 1 4 24 1 4 24 Liver 41.4 ± 1.3 48.8 ± 4.3 54.3 ± 11 557.9 ± 36.5 610.8 ± 115.7 805.1±240.2 Lung N.D.* 2.4 ± 1.3 2.3 ± 0.6 N.D. 30.3 ± 41.0 31.6±24.7 Spleen 92.2 ± 9.4 148.5 ± 17.2 138.7 ± 29.2 884.7 ± 182.9 755.9 ± 184.3 1454±225.9 Kidney 5.5 ± 1.0 9.3 ± 0.6 14.0 ± 1.9 N.D. N.D. 36.24±13.5 Muscle N.D. N.D. N.D. N.D. N.D. N.D. Antisense ws administered day 17 post tumor cell inoculation, with results expressed as µg per g tissue, following I.V. injection of liposomal VEGF-ASO at an antisense dose of 10 mg/kg (Mean ± standard error of the mean). *N.D. Below detectable limits which were set a a dpm value 2 times greater than background. www.landesbioscience.com Cancer Biology & Therapy 201 © Landes Bioscience 2004. Not for distribution. ANTISENSE PK IN THE MDA435/LCC6 BREAST CANCER MODEL liposomal ASO and spleen levels are 7 to 10-fold greater, depending on the time point. The increase in spleen and liver delivery is due in part to the natural tendency of liposomes to accumulate in these organs, even when prepared with surface grafted PEGs. Second, the tissue level of ASO in the kidney is at least 5-fold lower when the ASO is given in the liposomal form, an observation that is consistent with the fact that liposomes do not readily accumulate in organs such as the kidney (liposomal lipid levels were below detectable limits at the 1 and 4 hour time points) (data not shown). Similarly, the muscle ASO and lipid levels are below detectable limits, a consequence of little or no liposome accumulation in muscle tissue. It can be concluded from the data in Table 2 that the distribution of ASO, when given in the liposomal formulation is dictated by the biodistribution behav- ior of the liposomes. It is worth noting that the ASO to lipid ratios, estimated on the basis of the biodistribution data in Table 2, are lower than those measured in the plasma compartment (see Fig. 3E) and suggest that the ASO is lost from the liposomes both during and after tissue localization. Interestingly, the ASO to lipid ratio in the kidney, (estimated at the 24 h time point to be 0.4), is almost 3-fold higher that that measured in the plasma compartment. This is consistent with the fact that the kidney is the primary organ for free ASO removal, even after the ASO has been released from the liposomes. One of the benefits of utilizing a tumor model where the cells grow intraperitoneally within ascites fluid is that it provides easy access to the tumor and host cell populations residing within the peritoneal cavity and this, in turn, facilitates analysis of drug delivery and of associated therapeutics effects. To assess antisense delivery to the peritoneal cavity, MDA435/LCC6 tumor bearing animals (prepared as described for the studies shown in Fig. 4) were given an i.v. injection of free or liposomal VEGF-ASO on day 17 post-tumor cell inoculation. At 1, 4 and 24 hours post antisense injection, peritoneal fluid and associated cells were recovered by peritoneal lavage, as described in the Methods. There are two important conclusions that can be made on the basis of the data in Figure 5. First, the levels of ASO in the cell-free peritoneal fluid are comparable regardless of whether ASO has Figure 5. Peritoneal fluid (A) and peritoneal cell-associated (B) antisense lev- been given as a free or liposomal formulation. This result was surprising els following administration of free or liposomal VEGF-ASO at an antisense considering that there is a substantial increase in the blood levels and circu- dose of 10 mg/kg into female SCID/Rag2m MDA435/LCC6 ascites lation lifetime for the liposomal formulation, but are consistent with the tumor-bearing mice. Antisense was administered day 17 post tumor cell observations of others that the use of surface grafted PEGs can actually inoculation. (∇) liposomal ASO; (") free ASO. Results are the mean of 4 ± standard error of the mean. decrease extravasation rates and extent. For conventional small molecule anticancer drugs delivery in a PEG-free liposomal carrier typically results The data in Figure 4A is comparable to the plasma elimination data obtained in substantial increases in peritoneal cavity delivery, both in ascites tumor bearing and tumor free animals. It should be noted that the level of delivery in tumor free mice, suggesting that the presence of an established tumor did not impact the elimination of either the free or liposomal ASO. In addition, observed in the MDA435/LCC6 model is quite low, with as little as 1% of the injected dose accessing the peritoneal cavity at 24 h. The second impor- the data in Figure 4 suggests that the plasma elimination behavior of both ASO sequences are comparable, even though the HER-2/neu targeted ASO tant conclusion made regarding the data in Figure 5 concerns the increase in peritoneal cell delivery achieved when using the liposomal formulation, a is a 15 nucleotide sequence and the VEGF targeted ASO is a 21 nucleotide sequence. As was the case for non-tumor-bearing mice, the encapsulated result that is surprising considering that the presence of surface grafted PEGs should interfere with cell delivery. Although the error in this analysis antisense exhibited a much greater plasma residence time, with 4 hour levels being approximately 50-fold higher for liposomal formulation as compared is substantial and significant differences are only notable at the 4 h time point (p < 0.05), it is evident that there is an apparent increase in cell delivery. to free. Mean AUC0-24 analysis of these data demonstrate that the liposomal formulation provides for a 10-fold increase in mean plasma AUC0-24 when As much as 3 to 5 times greater levels of ASO are associated with the isolated peritoneal cells following administration of the liposomal formulation. compared to the free ASO. The tissue distribution of ASO and liposomal lipid following i.v. admin- Approximately 2 x 10 cells are isolated from the peritoneal cavity under the conditions employed in these studies (Fig. 6A) and this is a 30-fold increase istration of the VEGF-ASO was also determined and the results have been summarized in Tables 1 (free ASO) and 2 (liposomal ASO). There are two in cellularity when compared to nontumor bearing mice (data not shown). This cell number dropped to 1.4 x 10 when mice were treated with unen- important conclusions that can be drawn on the basis of the data in Table 1. First, the highest tissue levels of ASO obtained after administration of the capsulated ASO targeted to HER-2/neu, and further to levels comparable to nontumor bearing mice following treatment with liposomally formulated unencapsulated form was in the kidney. This is consistent with previous studies [20] and is indicative of rapid renal clearance of this agent. Second, HER2-ASO (Fig. 6A). Under conditions where the tumor bearing animals have been treated with saline (control treatment groups), almost 40% of the the levels of ASO in the spleen and liver are equivalent on a weight basis and the levels within these tissues are reasonably constant over the 24 h time cells isolated from the peritoneal cavity are murine (i.e., nonhuman) as judged by flow cytometric analysis. The influx of murine cells, primarily course. Liver accumulation of the injected ASO accounts for less than 10% of the injected ASO dose, while the spleen accumulation accounts for less neutrophils, into the peritoneal cavity is probably a consequence of tumor growth and perhaps the associated production of VEGF. As shown in Figure than 1% of the injected dose. When comparing these data to the liposomal formulation (Table 2), there are several notable differences. First, liver and 6B, of the total number of cells contained within the peritoneal cavity of saline treated mice, only 66% are viable, and of those, approximately 60% spleen ASO levels are substantially higher when the ASO is given in lipo- somal formulation. Liver levels are almost 3-fold greater in animals given were of human origin. Following treatment with free HER2-ASO, the 202 Cancer Biology & Therapy 2004; Vol. 3 Issue 2 © Landes Bioscience 2004. Not for distribution. ANTISENSE PK IN THE MDA435/LCC6 BREAST CANCER MODEL VEGF and HER-2/neu are just two A B examples of the many new molecular targets being researched for novel anti- cancer therapeutics. The agents targeting these molecules are also novel, with agents such as recombinant DNA, ASOs and monoclonal antibodies becoming predominant. These new agents can often not be administered systemically without the use of a cosol- vent or carrier systems of some sort, such as polymers, lipid complexes or liposomes. As is true for drugs such as anti-fun- 37 38 gals, anti-bacterials and several anti- 39-42 cancer agents, various ASO are being developed as liposomal formulations in order to protect the therapeutic agent 43-45 from degradation. A commonly used modification of ASO is the chemical variation of the backbone, the most commonly used being that of the substi- tution of a sulfur atom for one of the nonbridging oxygen atoms in the phos- Figure 6. Tumor burden (A) and percent murine (green)/human (red) cells (B) in MDA435/LCC6 tumor bearing phodiester bond. This phosphorothioate female SCID/Rag2m mice following treatment with free H2A-ASO or liposomal H2A-ASO. Results are the bond increases the serum terminal mean ± standard error of the mean of a minimum of 4 animals. half-life of ASO from minutes, to up to 9 percentage of viable cells in the total count did not change significantly, nor hours, a result that is primarily a consequence of improved stability did the ratio of murine to human cells, however a striking reduction in the against nucleases within the circulation. Liposomes are advantageous number of viable human cells was noted following treatment with liposo- for the delivery of many therapeutics as they are able to extend the mally encapsulated HER2-ASO, with less than 5% of the total cell count circulation lifetime as well as engendering significant increases in the being made up of viable human cells, with the remaining being composed plasma concentration of the associated agent. The increased circula- of murine cells (Fig. 6B). This result demonstrated not only a reduction in tion lifetime and plasma concentration have been shown to effect the total number of cells, but also a specific reduction in the number of increases in drug accumulation at sites of disease, particularly to human tumor cells within the peritoneal cavities of these mice. those diseases which cause damage to the surrounding blood vessels. This is particularly true for sites of tumor growth where there is DISCUSSION induction of neovasculature and the release of factors, such as VEGF, which cause increases in blood vessel permeability to circulating An assessment of the potential clinical suitability of novel anticancer 31-33 macromolecules. Studies demonstrating the therapeutic activity therapeutics typically includes the determination of anti-tumor of ASOs in vivo have had to rely on prolonged drug administration, activity in preclinical animal models of cancer, most of which are therefore it is anticipated that liposomal formulations of ASO mouse based. Ideally, the anti-tumor response to the agent being should improve therapy by increasing delivery of the intact sequence tested should assist in predicting activity when used in a clinical setting. to sites of disease and, perhaps, by reducing the frequency of drug An associated correlate to this ideal is that the model used should administration. also mimic the human disease state or condition. The MDA435/ The pharmacokinetic benefits of administering ASOs in a lipo- LCC6 ascites tumor model is a direct derivative of the estrogen somal formulation have been illustrated well by the data presented receptor negative, invasive and metastatic MDA-MB-435 cell in this report. Where the mean plasma AUC is more then 34-35 0-24h line and has been characterized as such by isotype and karyotype 10-fold greater than that achieved following i.v. administration of analysis. It has been suggested that the coexpression of molecular free ASO and the biodistribution of the ASO is dictated by the markers in neoplastic transformation functions as a significant prog- biodistribution of the liposomal carrier. It is important to note that nostic correlate for recurrence, and that the number of coexpressed the method to prepare the liposomal formulation of ASO used here markers is directly related to this significance. Given these points, has been reported elsewhere and these formulations are currently we chose to characterize the protein expression of VEGF and in preclinical development in our laboratory and others. HER-2/neu. Many tumor cells express and secrete VEGF at high levels, with References 1. Gasparini G, Toi M, Gion M, Verderio P, Dittadi R, Hanatani M, et al. Prognostic signif- the effect of inducing the formation of new vasculature to promote icance of vascular endothelial growth factor protein in node-negative breast carcinoma. J tumor growth. 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Published: Feb 2, 2004

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