Novel anticancer polymeric conjugates of activated nucleoside analogues

Abstract

Inherent or therapy-induced drug resistance is a major clinical setback in cancer treatment. The extensive usage of cytotoxic nucleobases and nucleoside analogues in chemotherapy also results in the development of specific mechanisms of drug resistance, such as nucleoside transport or activation deficiencies. These drugs are prodrugs; and being converted into the active mono-, di-, and triphosphates inside cancer cells following administration, they affect nucleic acid synthesis, nucleotide metabolism, or sensitivity to apoptosis. Previously, we actively promoted the idea that the nanodelivery of active nucleotide species, e.g., 5'-triphosphates of nucleoside analogues, can enhance drug efficacy and reduce nonspecific toxicity. In this study, we report the development of a novel type of drug nanoformulations, polymeric conjugates of nucleoside analogues, which are capable of the efficient transport and sustained release of phosphorylated drugs. These drug conjugates have been synthesized, starting from cholesterol-modified mucoadhesive polyvinyl alcohol or biodegradable dextrin, by covalent attachment of nucleoside analogues through a tetraphosphate linker. Association of cholesterol moieties in aqueous media resulted in intramolecular polymer folding and the formation of small nanogel particles containing 0.5 mmol/g of a 5'-phosphorylated nucleoside analogue, e.g., 5-fluoro-2'-deoxyuridine (floxuridine, FdU), an active metabolite of anticancer drug 5-fluorouracyl (5-FU). The polymeric conjugates demonstrated rapid enzymatic release of floxuridine 5'-phosphate and much slower drug release under hydrolytic conditions (pH 1.0-7.4). Among the panel of cancer cell lines, all studied polymeric FdU-conjugates demonstrated an up to 50× increased cytotoxicity in human prostate cancer PC-3, breast cancer MCF-7, and MDA-MB-231 cells, and more than 100× higher efficacy against cytarabine-resistant human T-lymphoma (CEM/araC/8) and gemcitabine-resistant follicular lymphoma (RL7/G) cells as compared to free drugs. In the initial in vivo screening, both PC-3 and RL7/G subcutaneous tumor xenograft models showed enhanced sensitivity to sustained drug release from polymeric FdU-conjugate after peritumoral injections and significant tumor growth inhibition. All these data demonstrate a remarkable clinical potential of novel polymeric conjugates of phosphorylated nucleoside analogues, especially as new therapeutic agents against drug-resistant tumors.

Figure 1

Formation of compact nanogels from polymer drug conjugates

Figure 2

31P-NMR spectrum of polymeric conjugates, CPVA31-p₄FdU (A) and CDex9-p4FdU (B) (phosphorus signals a-e are described in the text)

Figure 3

Transmission electron microscopy (TEM) images of nanogels formed from polymer conjugates: (A) CPVA31-p₄FdU, (B) its spermine complex (Spe), (C) CDex9-p₄FdU, and (D) its spermine complex. Samples were stained with vanadate.

Figure 4

Enzymatic hydrolysis of polymeric conjugates by snake venom phosphodiesterase I (VPDE). (A) The enzyme hydrolyzes the P-O bond at α-phosphate group in nanogel conjugate resulting in nucleoside 5′-phosphate (2). (B) Ion-pair HPLC profiles of initial (a) and hydrolyzed (b) CPVA31-p₄FdU. (C) Ion-pair HPLC profiles of initial (a) and hydrolyzed (b) CDex9-p₄T after 24 h-incubation with 0.01 units of VPDE enzyme.

Figure 5

In vitro drug release from polymeric conjugates CPVA31-p₄FdU (A) and CDex9-p₄FdU (B) at different pH in buffered saline.

Figure 6

Cytotoxicity of polymeric conjugates in drug-resistant human T-lymphoma CEM/araC/8 cells (A–C) and prostate carcinoma PC-3 cells (D).

Figure 7

Tumor growth inhibition in mice with subcutaneous human prostate carcinoma PC-3 (A) and gemcitabine-resistant follicular lymphoma RL7/G (B) tumors following the peritumoral injections of the polymeric conjugate CPVA31-p4FdU (dose 80 mg/kg, or 10 mg FdU/kg). The data were statistically significant with P<0.05 between (a) control and (b) treatment groups. (C) Tumor photographs in the end of the experiment B taken from control and treatment groups.

Scheme 1

Synthetic steps in the preparation of CPVA conjugates

Scheme 2

Synthesis of CDex-conjugates (compounds 1–11, see Scheme 1).

Scheme 3

Mechanism of acidic hydrolysis of polymeric conjugates and release of phosphorylated nucleosides (R = cholesterol).