Synthesis and therapeutic effect of styrene-maleic acid copolymer-conjugated pirarubicin

Abstract

Previously, we prepared a pirarubicin (THP)-encapsulated micellar drug using styrene-maleic acid copolymer (SMA) as the drug carrier, in which active THP was non-covalently encapsulated. We have now developed covalently conjugated SMA-THP (SMA-THP conjugate) for further investigation toward clinical development, because covalently linked polymer-drug conjugates are known to be more stable in circulation than drug-encapsulated micelles. The SMA-THP conjugate also formed micelles and showed albumin binding capacity in aqueous solution, which suggested that this conjugate behaved as a macromolecule during blood circulation. Consequently, SMA-THP conjugate showed significantly prolonged circulation time compared to free THP and high tumor-targeting efficiency by the enhanced permeability and retention (EPR) effect. As a result, remarkable antitumor effect was achieved against two types of tumors in mice without apparent adverse effects. Significantly, metastatic lung tumor also showed the EPR effect, and this conjugate reduced metastatic tumor in the lung almost completely at 30 mg/kg once i.v. (less than one-fifth of the maximum tolerable dose). Although SMA-THP conjugate per se has little cytotoxicity in vitro (1/100 of free drug THP), tumor-targeted accumulation by the EPR effect ensures sufficient drug concentrations in tumor to produce an antitumor effect, whereas toxicity to normal tissues is much less. These findings suggest the potential of SMA-THP conjugate as a highly favorable candidate for anticancer nanomedicine with good stability and tumor-targeting properties in vivo.

Keywords: Antimetastatic property; SMA-THP conjugate; antitumor effect; enhanced permeability and retention effect; prolonged plasma half-life.

Figure 1

Synthesis of styrene–maleic acid copolymer (SMA)-conjugated pirarubicin (THP) (SMA-THP conjugate). Chemical structures and conjugation pathway. DMF, N,N-dimethylformamide.

Figure 2

Characterization of styrene–maleic acid copolymer (SMA)-conjugated pirarubicin (THP) (SMA-THP conjugate). (a) HPLC analyses. (b) Quantification of free amino group by trinitrobenzene sulfonic acid method. (c) Hydrodynamic diameter in PBS of SMA-THP conjugate by dynamic light scattering. (d, e) Fluorescence spectra of SMA-THP conjugate. (f, g) Size exclusion chromatography of SMA-THP conjugate in the presence of different concentrations of BSA by using Sephacryl S-200. The apparent molecular weight of SMA-THP conjugate in the presence/absence of BSA was calculated by the calibration curve based on partition coefficient (K_av) using molecular weight standard markers.

Figure 3

In vitro cytotoxicity and intracellular uptake of free pirarubicin (THP) and styrene–maleic acid copolymer (SMA)-conjugated THP (SMA-THP conjugate). Cytotoxicities of free THP and SMA-THP conjugate against HeLa cells (a) and colon 26 cells (b) were measured by MTT assay. Values are means ± SEM. (c) Intracellular uptake of free THP and SMA-THP conjugate in HeLa cells. Values are means ± SEM (n = 3). (d) HPLC analyses of free THP at 6 h (d-i), SMA-THP conjugate at 10 h (d-ii), and 40 h (d-iii) in HeLa cells after each treatment.

Figure 4

Pharmacokinetics of free pirarubicin (THP) and styrene–maleic acid copolymer (SMA)-conjugated THP (SMA-THP conjugate) after i.v. injection. (a) Blood levels of both drugs after injection in an S-180 tumor model. (b, c) Tissue distribution of free THP and/or SMA-THP conjugate after i.v. injection in an S-180 tumor model. (d) Relative tissue distribution at 24 h after i.v. injection of free THP and SMA-THP conjugate in healthy SD rats. (e) Comparison of tissue distribution of SMA-THP conjugate at 24 and 72 h after i.v. injection in SD rats. Values are mean ± SEM (n = 3). *P < 0.05.

Figure 5

Effect of styrene–maleic acid copolymer (SMA)-conjugated pirarubicin (THP) (SMA-THP conjugate) on growth and lung metastasis in a colon 26 tumor model. Values are means ± SEM (n = 4–5). (a) Antitumor effect. (b) Body weight change in mice after treatment. Vertical arrows indicate injection time of drugs. Figure key shown in (a) also applies to (b). (c) Lung specimens in colon 26 tumor-bearing mice on day 51 after tumor inoculation. Arrows indicate metastatic tumors, quantified as shown in (d). *P < 0.05.

Figure 6

Antitumor effect of styrene–maleic acid copolymer (SMA)-conjugated pirarubicin (THP) (SMA-THP conjugate) against S-180 tumor. Vertical arrows indicate injection time of drugs. Values are means ± SEM (n = 5). (a) Antitumor effect. (b) Body weight change of ddY mice after treatment. (c) Survival rate of S-180 tumor-bearing ddY mice after treatment. Figure key shown in (a) also applies to (b) and (c). (d) Percent change of body weight of healthy normal male ddY mice after injection of different doses of SMA-THP conjugate.