hpma-docetaxel or gemcitabine conjugates and uses thereof

[0100] Example 1: Synthesis of HPMA-gemcitabine or docetaxel conjugates for drug delivery

[0101] HPMA comonomer drug conjugates were synthesized by polymerization of HPMA monomer and activated MA-GFLG-drug (SEQ ID NO: 1 ) comonomer in different molar ratios.

[0102] 1) Synthesis of HPMA comonomer

[0103] The synthesis of HPMA monomer was carried out as previously described (Kopecek and Bazilova, Eur. Polym. J., 9: 7-14 (1973)), as figure 1 shown. The MA-GG-ONp comonomer was prepared by a modified multistep reaction (Kopecek et al., Ann. N Y Acad. Sci., 446:93-104 (1985)).

[0104] plan 1

[0105]

[0106] To a solution of 1-amino-2propanol (65.6ml, 0.84mol) in 250ml of acetonitrile was added dropwise freshly distilled methacryloyl chloride (MACl) (41ml, 0.42mol, in 20ml of acetonitrile) at -5°C with vigorous stirring . A small amount of the inhibitor tert-octylcatechol was added to the solution. The reaction mixture was stirred at room temperature for an additional ...

[0148] Embodiment 2: biological test

[0149] 1) Growth of cancer cell lines

[0150] Cancer cell lines used to assay the effects of HPMA-drug conjugates were obtained from the following sources: human MDA-MB-231 (breast), HCT116 (colon) and PANC-1 (pancreas) from the American Standard Culture Center (ATCC) ( Manassas, VA). UMRC2 (kidney) was from the National Cancer Institute (Bethesda, MD). Cells were maintained in Dulbecco's Modified Eagle's Medium ("DMEM", Invitrogen) supplemented with 10% FBS, P / S and 10 mM HEPES. All cells were incubated at 37°C in humidified 5% CO 2 incubate.

[0151] 2) In vitro cell proliferation assay for human tumor cell lines

[0152] Growth inhibition assays of HPMA-drug conjugates against human cancer cell lines were performed using the sulforhodamine B ("SRB") method (Skehan et al., J. National Cancer Institute, 82: 1107-1112 (1990)). Briefly, exponentially growing cancer cells were treated with 2-3x10 3 Cells / well were seeded in 96-well ...

[0159] Example 3: Xenotransplantation Studies

[0160] To observe tumor growth inhibition in animal models, HPMA-conjugated docetaxel or gemcitabine was used on nude mouse xenograft models as described below.

[0161] Mia-Paca human pancreatic cancer cell or HCT116 cell suspension (3x10 6 cells) were subcutaneously injected into the lower flank of six-week-old female FoxN1 nude mice. When the tumor reaches an appropriate size (eg, about 50-60mm 3 volume), the mice were injected intravenously with phosphate-buffered saline (PBS) alone or the drug conjugate of the invention every 4-5 days. Animals are monitored for several weeks until control tumors reach, eg, about 1 cm in diameter, at which point animals are euthanized. Tumor size and tumor composition were determined and reported as fold change in tumor size ( Figure 1-Figure 3 ).

[0162] Using the model described in this example, it was shown that drug conjugates of the invention, such as those described in Example 1,...