Cancer Therapy: Preclinical
`
`Influence of Formulation Vehicle on Metronomic Taxane
`
`Chemotherapy: Albumin-Bound versus Cremophor
`
`EL— Based Paclitaxel
`
`Sylvia SW. Ng,1 Alex Sparreboom,2 Yuval Shaked,1 Christina Lee,1Shan Man,1 Neil Desai,3
`Patrick Soon—Shiong,3 William D. Figg,2 and Robert S. Kerbel1
`
`Abstract
`
`Purpose: Low-dose metronomic chemotherapy treatments, especially when combined with
`dedicated'antiangiogenic agents, can induce significant antitumor activity without serious toxic-
`ity in various preclinical models. It remains unclear, however, whether some cytotoxic drugs are
`better suited for metronomic regimens than others. Paclitaxel appears to be a strong candidate for
`metronomic chemotherapy given its ability to inhibit endothelial cell functions relevant to angio—
`genesis in vitro at extraordinarily low concentrations and broad-spectrum antitumor activity. Clin-
`ically relevant concentrations of the formulation vehicle cremophor EL in Taxol, however, were
`previously reported to nullify the antiangiogenic effect of paclitaxel, the result of which would
`hamper its usefulness in metronomic regimens. We hypothesized that ABI—OO7, a cremophor
`EL—free, albumin—bound, 130-nm form of paclitaxel, could potentially alleviate this problem.
`Experimental DesignzThe antiangiogenic activity of ABl—007 was assessed by multiple in vitro
`assays. The in vivo optimal dose of ARI-007 for metronomic chemotherapy was determined by
`measuring circulating endothelial progenitors in peripheral blood. The antitumor effects of
`metronomic and maximum tolerated dose ABI-OO7 and Taxol were then evaluated and compared
`in severe combined immunodeficient mice bearing human MDA—MD—231 breast cancer and PC3
`prostate cancer xenografts.
`Results: ABI-OO7 significantly inhibited rat aortic microvessel outgrowth, human endothelial cell
`proliferation, and tube formation. The optimal metronomic dose of ABl—OO7 was determined to be
`between 3 and 10 mg/kg. Metronomic ABI-OO7 but not Taxol, significantly suppressed
`tumor growth in both xenograft models. Furthermore, the antitumor effect of minimally toxic
`metronomic ABl—007 approximated that of the maximum tolerated dose ofTaxol.
`Conclusions: Our results underscore the influence of formulation vehicles on the selection of
`
`cytotoxic drugs for metronomic chemotherapy.
`
`An alternative dosing regimen to pulsatile maximum tolerated
`dose (MTD) or ”dose dense” and dose-intensive chemotherapy
`is ”metronomic chemotherapy": the frequent administration of
`such drugs at close regular intervals with no prolonged breaks
`
`Authors' Affiliations: 1Molecular and Cellular Biology Research, Sunnybrook
`Health Sciences Centre,Toronto, Ontario, Canada; 2Clinical Pharmacology
`Research Core, Medical Oncology Clinical Research Unit, National Cancer Institute,
`NIH, Bethesda, Maryland; and 3Abraxis BioScience, Santa Monica, California
`Received 12/16/05; revised 4/21/06; accepted 5/10/06.
`Grant support: National Cancer Institute of Canada (NCIC), Canadian Institutes of
`Health Research (RS. Kerbel) and Terry Fox Research Grant for New Investigators
`funded through the NCIC.
`The costs of publication of this article were defrayed in part by the payment of page
`charges. This article must therefore be hereby marked advertisement in accordance
`with 18 U.S,C. Section 1734 solely to indicate this fact.
`Note: S.S.W. Ng and Y. Shaked are recipients of postdoctoral fellowships from the
`Canadian Institutes of Health Research.
`Requests for reprints: Robert S. Kerbel, Molecular and Cellular Biology Research,
`Sunnybrook Health Sciences Centre, Room 8-217, 2075 BayviewAvenue,Toronto,
`Ontario, Canada, M4N 3M5. Phone: 416-480-5711; Fax: 416-480-5884; E-mail:
`Robert.Kerbel@sri.utoronto.ca.
`©2006 American Association for Cancer Research.
`doi:10.1158/1078-0432.CCR-05-2762
`
`long periods of time (1). The reduced toxicity and
`over
`comparable or even increased efficacy of metronomic regimens
`compared with some MTD counterparts have been shown in a
`number of preclinical models (2, 3). In addition, metronomic
`chemotherapy regimens are particularly well suited for long—
`term combination with relatively non —toxic—targeted biological
`therapeutics especially antiangiogenic drugs (4, 5), sometimes
`being used after an initial short course of MTD chemotherapy
`(i.e., ”chemo-switching” protocols; refs. 4, 6). Some phase II
`clinical
`trials have been reported with encouraging results,
`both in terms of antitumor efficacy and reduced toxicity,
`although such results need to be validated in larger
`randomized phase II or III
`trials (7—9). These trials have
`involved combinations of two metronomically given drugs
`[e.g., cyclophosphamide and methotrexate (7) or cyclophos—
`phamide and- etoposide (8)] given orally on a daily basis, or
`similar protocols in combination with a drug such as
`bevacizumab (Avastin),
`the anti-vascular endothelial growth
`factor antibody (9).
`Virtually every class of chemotherapeutic drug has been
`reported to have antiangiogenic properties (10), which in many
`cases can be amplified by metronomic dosing schedules (1).
`
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`Cancer Therapy: Preclinical
`
`However, the selection of chemotherapeutic drugs for metro-
`nomic regimens remains somewhat arbitrary. It is not clear
`whether some agents or classes of agents are better suited for
`metronomic chemotherapy than others. Taxanes, such as
`paclitaxel, would seem to be excellent candidates based on
`the finding that ultra low (e.g., picomolar) concentrations of
`paclitaxel can selectively inhibit endothelial functions relevant
`to angiogenesis, or even kill such cells ( 11 —15). In addition,
`long-term metronomic chemotherapy using microtubule-
`inhibiting taxanes as opposed to mutagenic and hence
`potentially carcinogenic DNA damaging drugs, such as alkylat-
`ing agents, could also be an advantage, especially in patients
`receiving adjuvant metronomic therapy regimens over long
`periods of time for early-stage disease ( 16). Furthermore, taxane
`metronomic chemotherapy may be useful to combine with
`metronomic chemotherapy using another class of drug, such as
`anti—metabolites (e.g., UFT, the oral 5-fluorouracil prodrug; ref.
`16). For example, we have recently reported that a concurrent
`combination of daily oral low-dose UFI‘ and cyclophospha-
`mide can successfully control highly advanced visceral metas-
`tases of human breast cancer in immunodeficient mice,
`whereas UFF or cyclophosphamide used alone could not
`(17). However, clinically relevant concentrations of the
`formulation vehicle cremophor EL in Taxol were previously
`reported to nullify the antiangiogenic activity of paclitaxel,
`suggesting that this agent or other anticancer drugs formulated
`in cremophor EL and other commonly used solubilization
`vehicles, such as polysorbate 80 (Tween 80), may need to be
`used at much higher doses than anticipated to achieve effective
`metronomic chemotherapy (18). As such,
`the advantage of
`reduced acute serious side effects associated with low-dose
`
`paclitaxel regimens versus conventional MTD paclitaxel may be
`compromised. Clearly, the presence of cremophor EL in Taxol
`hampers the use of paclitaxel in metronomic chemotherapy.
`This may explain, for example, the results of Klement et al.
`(19), in which metronomic Taxol on its own had little obvious
`effects on transplanted primary human breast tumors in several
`models.
`
`ABI-007 (Abraxane), a novel cremophor EL—free, albumin-
`bound, 130-nm form of paclitaxel, was developed to retain
`the therapeutic benefits of paclitaxel but eliminate cremophor
`EL—associated toxicities in the Taxol formulation. Several
`
`clinical trials have shown the improved pharmacokinetic and
`toxicity profiles as well as therapeutic efficacy of ABI-007 over
`Taxol in MTD regimens (20—24). We hypothesized that the
`cremophor EL-free nature of ABl-007 may render paclitaxel-
`based metronomic chemotherapy feasible. With this in mind,
`the primary objective of this study was to evaluate the
`therapeutic potential of paclitaxel-based metronomic regimens
`using ABI-007.
`
`Materials and Methods
`
`injection containing paclitaxel at 6 mg/mL in a
`Drugs. Taxol
`mixture of cremophor EL and ethanol USP (1:1 v/v; Bristol-Myers
`Squibb Canada, Montreal, Canada) was purchased from the local
`hospital pharmacy. ARI-007 was obtained from American BioScience
`(Santa Monica, CA).
`Rat aortic ring assay. Twelve—well tissue culture plates were coated
`with 250 uL of Matrigel (Collaborative Biomedical Products, Bedford,
`
`MA) and allowed to gel for 30 minutes at 37°C and 5% C02. Thoracic
`aortas were excised from 8— to 10-week-old male Sprague-Dawley rats.
`Following removal of fibroadipose tissues, the aortas were cut into
`l-mm-long cross-sections, placed on Matrigel-coated wells, and covered
`with an additional 250 uL of Matrigel. After the second layer of Matrigel
`had set, the rings were covered with EGM-II and incubated overnight at
`37°C and 5% C02. EGM-II consists of endothelial cell basal medium
`(EBM-II; Cambrex, Walkersville, MD) plus endothelial cell growth
`factors provided as the EGM-II Bulletkit
`(Cambrex). The culture
`medium was subsequently changed to EBM-II supplemented with 2%
`fetal bovine serum, 0.25 ug/mL amphotericin B, and 10 jig/ml.
`gentamicin. Aortic rings were treated with EBM-II containing the
`vehicle (0.9% saline/albumin), carboxyamidotriazole (12 ug/mL), or
`ABI-OO? (0.05-10 nmol/L paclitaxel) for 4 days and photographed on
`the fifth day using a X2.5 objective. Carboxyamidotriazole, a known
`antiangiogenic agent, was used at higher than clinically achievable
`concentration as positive control (25). Experiments were repeated four
`times using aortas from four different rats. The area of angiogenic
`sprouting,
`reported in square pixels, was quantified using Adobe
`Photoshop 6.0.
`Endothelial cell proliferation assay. Human umbilical vein endo-
`thelial cells (HUVEC; Cambrex) were maintained in EGM-II at 37°C
`and 5% C02. HUVECs were seeded onto 12-well plates at a density of
`30,000 per well and allowed to attach overnight. The culture medium
`was then aspirated, and fresh culture medium containing either the
`vehicle (0.9% saline/albumin), or ARI-007 (0.05-10 nmol/L paclitaxel)
`was added to each well. After 48 hours, cells were trypsinized and
`counted with a Coulter Z1 counter (Coulter Corp., Hialeah, FL). All
`experiments were repeated thrice.
`Endothelial cell
`tube formation assay. Eight-well slide chambers
`were coated with 150 11L of Matrigel and allowed to gel at 37°C and 5%
`C02 for 30 minutes. HUVECs were then seeded at 30,000 per well in
`EGM-II containing either the vehicle (0.9% saline/albumin) or ABI-007
`(0.05-10 nmol/L paclitaxel) and incubated at 37°C and 5% C02 for 16
`hours. After incubation, slides were washed in PBS, fixed in 100%
`methanol for 10 seconds, and stained with Difouick solution II (Dade
`Behring, Inc., Newark, DE) for 2 minutes. To analyze tube formation,
`each well was digitally photographed using a X2.5 objective. A
`threshold level was set to mask the stained tubes. The corresponding
`area was measured as the number of pixels using MetaMorph software
`(Universal Imaging, Downingtown, PA). Experiments were repeated
`thrice.
`
`Determination of the in vivo optimal biological dose of ABI-007 by
`measuring circulating endothelial cells and circulating endothelial
`progenitors.
`Six- to 8-week—old female BALB/c] mice were random-
`ized into the following eight groups (n = 5 each): untreated, treated
`with i.p. bolus injections of either the drug vehicle (0.9% saline/
`albumin), or ABI-OO7 at 1, 3, 6, 10, 15, or 30 mg/kg paclitaxel daily
`for 7 days. At the end of the treatment period, blood samples were
`drawn by cardiac puncture and collected in EDTA-containing
`vacutainer tubes (Becton Dickinson, Franklin Lakes, NI). Circulating
`endothelial cells (CBC) and circulating endothelial progenitors (CEP)
`were enumerated using four-color
`flow cytometry as previously
`described (3, 26). Monoclonal antibodies specific for CD45 were
`used to exclude CD45+ hematopoietic cells. CECs and their CEP
`subset were depicted using the murine endothelial markers fetal liver
`kinase l/vascular endothelial growth factor receptor 2, CD13, and
`CD117 (BD PharMingen, San Diego, CA). Nuclear staining (Pro-
`count; BD Biosciences, San Jose, CA) was done to exclude the
`possibility of platelets or cellular debris interfering with the accuracy
`of CBC and CEP enumeration (27, 28). After red cell
`lysis, cell
`suspensions were evaluated by a FACSCalibur (BD Biosciences) using
`analysis gates designed to exclude dead cells, platelets, and debris. At
`least 100,000 events per sample were obtained to analyze the
`percentage of CECs and CEPs. The absolute number of CECs and
`CEPs was then calculated as the percentage of the events collected in
`the CBC and CEP enumeration gates multiplied by the total white
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`Metronomic Chemotherapy with Albumin-Bound Paclitaxel
`
`Statistics. All results were presented as mean i SE. Comparisons
`were made with one—way ANOVA followed by Student Newman-Keuls
`or Dunnett’s test with P < 0.05 as the criterion for statistical
`significance. Correlation between viable CEP levels in peripheral blood
`and intratumoral microvessel density in the human tumor xenografts
`studies was examined using the nonparametric Spearman test with the
`level of significance set at P < 0.05.
`
`Results
`
`Rat aortic angiogenesis, HUVEC proliferation, and tube
`formation in response to ARI-007. We first asked whether the
`antiangiogenic effects of paclitaxel can be effectively delivered
`by the cremophor EL—free ABI-007 in vitro. As shown in
`Fig. 1A, ABI-007 suppressed aortic microvessel outgrowth in a
`
` A
`
`280000
`
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`
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`
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`80000
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`
`Meanpixeldensity
`
`.
`Control CAI
`
`0.05
`
`.
`5
`
`10
`
`0.5
`
`1
`ABI—007 (nM)
`Treatment
`
`
`
`
`
`”/0cellsrelativetovehiclecontrol
`
`1 20
`
`l 00
`
`80
`
`60
`
`40
`
`20
`
`0
`
`st
`
`i'
`
`Control
`
`
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`
`ABI—007 (nM)
`Treatment
`
`
`
`160000
`
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`120000
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`80000
`60000
`40000
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`Control
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`0.05
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`5
`
`10
`
`0.5
`
`l
`ABl-OO7 (nM)
`Treatment
`
`
`
`
`
`Fig. 1. Effects of ABI-007 on rat aortic ring angiogenesis (A), HUVEC proliferation
`(B), and tube formation (C). Columns, mean; bars, SE. ', P ( 0.05, significantly
`different from the vehicle control. CAI, carboxyamidotriazole.
`
`cell count. Percentages of stained cells were determined and
`compared with the appropriate negative controls. Positive staining
`was defined as being greater than nonspecific background staining. 7-
`Amino—actinomycin D was used to enumerate viable versus apoptotic
`and dead cells (29).
`Human tumor xenograft therapy studies. Human prostate cancer cell
`line PC3 and human breast cancer cell
`line MBA-MD-231 were
`
`obtained from the American Type Culture Collection (Manassas, VA)
`and maintained at 37°C and 5% C02 in RPMI 1640 supplemented
`with 10% fetal bovine serum and antibiotics (100 units/mL penicillin
`and 100 ug/mL streptomycin). All animal experiments were done in
`accordance with institutional guidelines for animal welfare. PC3 (5 X
`106 cells) were injected so into 6- to 8-week-old male severe combined
`immunodeficient mice, whereas MDA-MB-231 (2 X 106 cells) were
`implanted orthotopically into the mammary fat pad of female severe
`combined immunodeficient mice. When the primary tumor volume
`reached ~ 150 to 200 mm3, animals were randomized into eight
`groups (n = 5-10 per group). Each group was treated with either 0.9%
`saline/albumin vehicle control, cremophor EL vehicle control, metro-
`nomic Taxol (1.3 mg/kg, i.p., qd), metronomic ABI-007 (3, 6, or 10
`mg/kg paclitaxel, i.p., qd), MTD Taxol (13 mg/kg, i.v., qu5, 1 cycle),
`or MTD ABI—007 (30 mg/kg paclitaxel, i.v., qu5, 1 cycle). Perpendic-
`ular tumor diameters were measured with a caliper once a week, and
`their volumes were calculated using the formula 11/6 X a X b2, where a
`is the longest dimension of the tumor, and b is the width. At the end of
`the treatment period, blood samples were drawn by cardiac puncture
`from mice in all groups and collected in EDTA—containing vacutainer
`tubes (Becton Dickinson). CECs and CEPs were enumerated as
`described above. Tumors were harvested, snap frozen in optimum
`cutting temperature compound (Sakura Finetek USA, Inc., Torrance,
`CA) in liquid nitrogen and subsequently processed for immunofluo-
`rescence staining.
`Five-
`Detection and quantification ofintratumoral microvessel density.
`micrometer-thick sections obtained from each frozen tumor were
`
`stained with H&1E for histologic examination. For detection of micro-
`vessels, sections were stained with a rat anti-mouse CD31/platelet/
`endothelial cell adhesion molecule 1 antibody (111,000; BD PharMin—
`gen) followed by a Texas Red—conjugated goat anti-rat secondary
`antibody (1:200; Jackson ImmunoResearch Laboratories, Inc., West
`Grove, PA). A single microvessel was defined as a discrete cluster or
`single cell stained positive for CD31/platelet/endothelial cell adhesion
`molecule 1, and the presence of a lumen was not required for scoring as
`a microvessel. The microvessel density for each tumor was expressed as
`the average count of the three most densely stained fields identified
`with a X20 objective on a Zeiss AxioVision 3.0 fluorescence microscopic
`imaging system. Four to five different tumors per each vehicle control or
`treatment group were analyzed.
`In vivo angiogenesis evaluation. The Matrigel plug perfusion assay
`was done with minor modifications as previously described (4). Briefly,
`0.5 mL Matrigel supplemented with 500 ng/mL of basic fibroblast
`growth factor (R&D Systems, Inc., Minneapolis, MN) was injected so
`on day 0 into the flanks of 10-week-old female BALB/c] mice. On day 3,
`animals were randomly assigned to eight groups (n = 5 each). Each
`group was treated with either 0.9% saline/albumin vehicle control,
`cremophor EL vehicle control, metronomic Taxol (1.3 mg/kg, i.p., qd),
`metronomic ABl—007 (3, 6, or 10 mg/kg paclitaxel, i.p., qd), MTD Taxol
`(13 mg/kg,
`i.v., quS), or MTD ABI-007 (30 mg/kg paclitaxel,
`i.v.,
`quS). As a negative control, five additional female BALB/cl mice of
`similar age were injected with Matrigel alone. On day 10, all animals
`were injected iv. with 0.2 mL of 25 mg/mL FlTC—dextran (Sigma, St.
`Louis, MO). Plasma samples were subsequently collected. Matrigel
`plugs were removed, incubated with Dispase (Collaborative Biomedical
`Products, Bedford, MA) overnight at 37°C, and then homogenized.
`Fluorescence readings were obtained using a FL600 fluorescence plate
`reader (Biotech Instruments, Winooski, VT). Angiogenic response was
`expressed as the ratio of Matrigel plug fluorescence to plasma
`fluorescence.
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`Cancer Therapy: Preclinical
`
`concentration-dependent manner relative to the vehicle con—
`trol,
`reaching statistical significance at 5 nmol/L (53%
`inhibition) and 10 nmol/L (68% inhibition). The amount of
`albumin present at each concentration of ABI-OO7 alone did
`not inhibit angiogenesis (data not shown).
`HUVEC proliferation was significantly inhibited by ABI-007
`at 5 and 10 nmol/L by 36% and 41%, respectively. ABI-007
`also blocked tube formation by 76% at both 5 and 10 nmol/L
`(Fig. 1B and C).
`In vivo optimal biological dose for metronomic ABI-
`007. Next,
`the optimal dose of ABI-007 to be used in
`metmomic chemotherapy was determined. Enumeration of
`viable CEPs can be used as a surrogate pharmacodynamic
`marker to establish the optimal biological dose for targeted
`antiangiogenic drugs (26) and metronomic chemotherapy
`(30). Figure 2 shows that ABl-007 given i.p. daily for 7 days
`at 3 and 10 to 30 mg/kg significantly decreased CEP levels in
`non—tumor-bearing BALB/c] mice. However, ABI-007 at 10 to
`30 mg/kg was associated with a significant reduction of white
`blood cell count
`indicative of toxicity (data not shown).
`Although the reduction of CEP levels by ABI-007 at 6 mg/kg did
`not reach statistical significance, decrease in white blood cell
`count was not evident. We, therefore, concluded that the in vivo
`optimal biological dose for metronomic ABI-007 was between
`3 and 10 mg/kg. In a preliminary study, metronomic Taxol at
`1.3, 3, 6, or 13 mg/kg given i.p. (qu7) did not significantly
`reduce viable CEP levels, whereas metronomic Taxol at 30 mg/
`kg or higher resulted in severe toxicity and eventually mortality
`in mice (data not shown). It was previously reported that the
`i.p. administration of Taxol at doses commonly used in the
`clinic resulted in entrapment of paclitaxel in cremophor EL
`micelles in the peritoneal cavity and, consequently, insignifi-
`cant plasma paditaxel concentration (31). This would explain
`why doses of metronomic Taxol (1.3, 3, 6, and 13 mg/kg) that
`did not cause death failed to change viable CEP levels. In this
`case, the i.p. administration of metronomic Taxol at 1.3 mg/kg
`would not be any different from that at 13 mg/kg. We,
`
`therefore, selected the lower dose (1.3 rug/kg) to minimize the
`amount of cremophor EL per paclitaxel administration for
`subsequent experiments.
`Antitumor efl'ects of metronomic and MTD ARI—007 versus
`Taxol. Metronomic ABI—007 (3, 6, and 10 mg/kg) but not
`Taxol (1.3 mg/kg) given i.p. daily for 4 weeks significantly
`inhibited growth of both MDA—MB-231 and PC3 tumors
`(Fig. 3A and B). Neither ABI-007 nor Taxol given metronom-
`ically induced weight loss in mice (Fig. 3C and D). Although
`MTD ABI—007 (30 mg/kg) suppressed tumor growth more
`effectively than MTD Taxol (13 mg/kg), significant weight loss
`was noted with the former, indicating toxicity (Fig. 3C and D).
`In addition,
`two of five mice treated with MTD ARI—007
`displayed signs of paralysis in one limb 6 days after the last
`dose of drug. The paralysis was transient and resolved within 24
`to 48 hours. Interestingly, the antitumor effect of metronomic
`ABI-007 at 6 mg/kg approximated that of MTD Taxol in the
`MDA—MB—231 xenograft model (Fig. 3A). Increasing the dose of
`metronomic ABI-007 to 10 mg/kg did not seem to confer more
`pronounced tumor growth inhibition (Fig. 3A). In contrast,
`metronomic ABI-007 elicited greater antitumor response at 10
`mg/kg than at 3 and 6 mg/kg in the PC3 xenografts (Fig. 3B).
`Viable CEP levels in response to metronomic and M’ID ABI-007
`versus Taxol. As illustrated in Fig. 4A, metronomic ABI-OO7
`significantly decreased the levels of viable CEPs in a dose-
`dependent manner in MDA-MB-231 tumor-bearing mice.
`Viable CEP levels also exhibited a dose-dependent reduction
`in response to metronomic ABI—007 in PC3 tumor-bearing mice
`but reaching statistical significance only at 10 mg/kg (Fig. 4B).
`Although metronomic Taxol seemed to reduce the levels of
`CEPs slightly in both xenograft models, the reduction did not
`reach statistical significance (Fig. 4A and B). Both MTD ABI-007
`and MTD Taxol significantly lowered viable CEPs compared
`with their respective vehicle controls in MDA—MB-231 tumor—
`bearing mice, whereas only MTD Taxol did so in the PC3
`tumor-bearing mice (Fig. 4A and B).
`Effects of metronomic and MTD ARI—007 and Taxol on
`intratumoral microvessel density.
`In MDA-MB-231 tumors,
`metronomic ABI-007 at 6 and 10 mg/kg as well as MTD ABI-
`007 seemed to reduce microvessel density slightly although
`statistical significance was not reached (Fig. 5A).
`In PC3
`tumors, metronomic ABl-007 at 3 and 10 mg/kg seemed to
`decrease microvessel density but without reachng statistical
`significance (Fig. 5A). Interestingly, a significant correlation
`existed between microvessel density and the levels of viable
`CEPs in the MDA—MB-231 (Fig. 5B; r = 0.76, P = 0.04) but not
`in the PC3 (Fig. 5C; r = —0.071, P = 0.88) model.
`In vivo antiangiogenic activity of metronomic and MTD ABI-
`007 versus Taxol.
`In the Matrigel plug perfusion assay,
`metronomic ABI-007 at 6 and 10 mg/kg seemed to decrease
`angiogenesis, although the inhibition did not reach statistical
`significance (Fig. 6). Angiogenesis seemed to be unaltered by
`metronomic ABI-007 at 3 mg/kg, MTD ARI-007, MTD, and
`metronomic Taxol relative to the respective vehicle controls
`(Fig. 6). These observations were similar to the intratumoral
`microvessel density results described above.
`
`Discussion
`
`Our results show that metronomic chemotherapy using
`albumin-bound nanoparticle paclitaxel
`(ABI-007), but not
`
`Treatment
`
`an
`(.1.
`
`3“D.
`u.)
`
`D2 £
`
`06>
`“:
`.0as
`I0.D
`EZ .0N
`
`.0o
`
`ARI-007 (mg/kg)
`
`Fig. 2. Determination of optimal biologic dose of ABl-007 for metronomic dosing.
`Levels of viable CEPs in peripheral blood of BALB/cJ mice in response to escalating
`doses of ABl-007. Untr'd, untreated control; S/A, saline/albumin vehicle control.
`Columns, mean; bars, SE. ', P ( 0.05, significantly different from the untreated
`control.
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`Tumorvolume(mm3
`
`55
`
`-I— Saline/Albumin control
`+ Crcmophor EL control
`+ melronomic Taxol 1.3 mg/kg
`+ metronomic ARI—007 3 mg/kg
`+ merronomic ABl-OO7 6 mg/kg
`+ metronomic ARI-007 10 mg/kg
`—I— MTD Taxol quS
`+ MTD ARI-007 quS
`
`NOO0
`
`
`
`
`
`Tumorvolume(mm3
`
`Metronomic Chemotherapy with Albumin—Bound Pac/itaxe/
`
`
`
`+ Saline/Albumin control
`+ Cremophor EL control
`+metronomic Taxol 1.3 mg/kg
`+mclronomic ABI-007 3 mg/kg
`+metronomic ARI-007 6 mg/kg
`+mctronomic ARI-007 10 mg/kg
`—-I— MTD Taxol quS
`+MTD ARI-007 qu5
`
`NOCO._l_l_l_l
`
`I-
`
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`
`Starl TX on Day 21
`
`-
`
`0
`
`5
`
`10
`
`15
`
`20
`
`30
`25
`Day
`
`35
`
`4O
`
`45
`
`50
`
`55
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
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`50
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`55
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`
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`Mouseweight(g)
`
`
`
`Mouseweight(g)
`
`15
`
`20
`
`30
`25
`Day
`
`35
`
`40
`
`45
`
`50
`
`55
`
`15
`
`20
`
`30
`25
`Day
`
`35
`
`40
`
`45
`
`50
`
`Fig. 3. Effects of ABl-007 and Taxol used in metronomic or MTD regimens on MBA-MD-231 (A) and PC3 (B) tumor growth and the body weight of MDA-MB-231 (C) and
`PC3 (D) tumor-bearing SCID mice. Five to 10 mice were used per control or treatment group. Points, mean; bars, SE.
`
`paclitaxel formulated in cremophor EL (Taxol), exhibits potent
`in viva antitumor activity.
`It was previously reported that
`clinically relevant concentrations of formulation vehicles, such
`as cremophor EL, nullify the in vitro antiangiogenic effect of
`taxanes (18). Paclitaxel at 4 nmol/L dissolved in DMSO but not
`in cremophor EL suppressed rat aortic angiogenesis and
`HUVEC proliferation (18). In the present study, ABI-OO7 at 5
`nmol/ L induced responses similar to those elicited by paclitaxel
`at 4 nmol/L dissolved in DMSO, indicating that the antiangio-
`genic property of paclitaxel was effectively delivered by
`cremophor EL—free ABI-007. Desai et al. (32) recently showed
`that paclitaxel in ABI-007 is actively transported into and across
`endothelial cells by gp60 (a specific albumin receptor) —
`mediated caveolar transcytosis, a process that is inhibited by
`cremophor EL in Taxol.
`One cycle of either MTD ABI—007 or MTD Taxol was shown
`herein to cause marked tumor growth inhibition and transient
`regression in two different xenograft models (MDA—MB-231
`and PC3). However, MTD Taxol—treated tumors rapidly
`resumed growth 3 weeks after the last dose of drug (day 42),
`whereas the growth of MTD ABI-007 —treated tumors continued
`to be suppressed. The ability of MTD ABI-OO7 but not MTD
`
`to significantly reduce viable CEPs in MDA—MB—231
`Taxol
`tumor-bearing mice might contribute, at least in part, to the
`enduring antitumor effect of the former. A higher paclitaxel
`plasma clearance and a larger volume of distribution for ABI-
`007 than for Taxol was recently reported in humans (33) as
`well as more rapid cellular uptake and binding (32), suggesting
`that paclitaxel
`in the ABI-OO7 formulation might be more
`effective against CEPs than Taxol. Higher intratumoral pacli-
`taxel concentration achieved by the ABI-007 formulation could
`be another contributing factor (32, 33). We cannot explain why
`there was a lack of a significant decrease in viable CEPs by MTD
`ABI-OO7 in PC3 tumor—bearing mice. The possibility that the so
`PC3 tumors might be less dependent on recruitment of CEPs
`for angiogenesis compared with the orthotopic MDA—MB-231
`tumors cannot be excluded.
`
`The significant weight loss and transient paralysis associated
`with MTD ABI—007 treatment and the absence of cremophor EL
`in the formulation prompted us to explore the feasibility of
`using ABI-007 in metronomic regimens as a less toxic but still
`highly effective antitumor alternative, one that would be well
`suited for long-term combination with other drugs, such as
`vascular endothelial growth factor—targeted antiangiogenic
`
`www.aacrjourna|s.org
`
`4335
`
`Clin Cancer Res 2006;12(14) July15, 2006
`
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`5
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`

`total dose of ABI-007 given over 4 weeks in the metronomic
`regimen, especially in the 10 mg/kg/d treatment group, was
`substantially higher than that given over 5 days in the MTD
`regimen (280 versus 150 mg), and yet no significant weight loss
`was evident in the former. In marked contrast, metronomic
`Taxol failed to suppress tumor growth or significantly alter
`viable CEP levels. In all likelihood, this can be attributed to the
`entrapment of paclitaxel
`in cremophor micelles in the
`
`>
`
`- MDA-MB-231
`|:I PC3
`
`
`
`
`
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`
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`30
`Microvessel count in ZOOX field
`
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`
`
`
`30
`Microvessel count in 200X field
`
`lntratumoral microvessel density (A) of MDA-MB-231 (l) and P63 (El)
`Fig. 5.
`xenografts treated with (A) saline/albumin control; (B) cremophor EL control; (C)
`metronomic 1.3 mg/kgTaxol; (D, E, and F) metronomic 3,6, and 10 mg/kg ABl-007,
`respectively; (G) MTD Taxol; and (H) MTD ABl-007. Columns, mean; bars, SE.
`Correlation between intratumoral microvessel density and the number of viable
`CEPs in peripheral blood in MDA-MB-231 (B) and PC3 (C) tumor-bearing mice.
`
`Cancer Therapy: Preclinical
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`Fig. 4. Changes in the levels of viable CEPs in peripheral blood of MDA-MBZ31
`(A) and PC3 (B) tumor-bearing severe combined immunodeficient mice after
`treatment with metronomic or MTD ABl-007 and Taxol. Columns, mean; bars, SE.
`a, P ( 0.05, significantly different from saline/albumin vehicle control. b, P < 0.05,
`significantly different from cremophor EL vehicle control.
`
`drugs (4, 6). Naturally, the selection of optimal doses of ABI-
`007 for such regimen is pivotal
`to achieving therapeutic
`efficacy. We have recently shown the potential use of viable
`CEP levels in peripheral blood as a biomarker to determine the
`optimal biological doses of antiangiogenic drugs (26) and for
`metronomic chemotherapy, at least in mice (30). For example,
`the dose of DC101 (800 pg), a rat monoclonal antibody against
`mouse vascular endothelial growth factor receptor 2, which
`induced the lowest
`levels of viable CBPs also elicited the
`
`greatest reduction in tumor volume, whereas dose escalation up
`to 2,000 pg failed to decrease both variables any further (26). In
`the current study, metronomic ABI-007 at 3 and 10 to 30 mg/kg
`significantly reduced viable CEP levels in non—tumor—bearing
`BALB/c] mice. However, ABI-007 at 10 to 30 mg/kg was also
`associated with significant reduction in white blood cell count,
`indicative of toxicity. We, therefore, deduced that the optimal
`biological doses for metronomic ABI-007 were between 3 and
`10 mg/kg. Indeed, metronomic ABI—OO7 at 3 to 10 mg/kg given
`i.p. daily for 4 weeks effectively inhibited MDA—MB-231 and
`PC3 tumor growth. The observed decrease in tumor volume
`was accompanied by a dose-dependent reduction in viable CEP
`levels in both xenograft models. It should also be noted that the
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`
`6
`
`

`

`Metronomic Chemotherapy with Albumin-Bound Paclitaxel
`
`metronomic ABI-007 doses was evident, whereas MTD Taxol
`and MTD ABI-007 did