Poly(ethylene glycol)-block-poly(d,l-lactic acid) micelles containing oligo(lactic acid)8-paclitaxel prodrug: In Vivo conversion and antitumor efficacy

Abstract

Poly(ethylene glycol)-block-poly(d,l-lactic acid) (PEG-b-PLA) micelles affect drug solubilization, and a paclitaxel (PTX) loaded-PEG-b-PLA micelle (PTX-PM) is approved for cancer treatment due to injection safety and dose escalation (Genexol-PM®) compared to Taxol®. However, PTX-PM is unstable in blood, has rapid clearance, and causes dose-limiting toxicity. We have synthesized a prodrug for PTX (7-OH), using oligo(lactic acid) as a novel pro-moiety (o(LA)₈-PTX) specifically for PEG-b-PLA micelles, gaining higher loading and slower release of o(LA)₈-PTX over PTX. Notably, o(LA)₈-PTX prodrug converts into PTX by a backbiting reaction in vitro, without requiring esterases. We hypothesize that o(LA)₈-PTX-loaded PEG-b-PLA micelles (o(LA)₈-PTX-PM) has a lower C_max and higher plasma AUC than PTX-PM for improved therapeutic effectiveness. In Sprague-Dawley rats at 10 mg/kg, compared to o(LA)₈-PTX-PM (10% w/w loading) and PTX-PM (10%), o(LA)₈-PTX-PM (50% w/w loading) produces a 2- and 3-fold higher plasma AUC_0-24 of PTX, lactic acid-PTX, and o(LA)₂-PTX (o(LA)_0-2-PTX), respectively. For o(LA)₈-PTX-PM at 10 and 50% w/w loading, PTX and lactic acid-PTX are major bioactive metabolites, respectively. Fast prodrug conversion of o(LA)₈-PTX in vivo versus in vitro (by backbiting) suggests that o(LA)₈ is a good substrate for esterases. At 60 mg/kg (qwx3), o(LA)₈-PTX-PM (50%) has higher antitumor activity than o(LA)₈-PTX-PM (10%) and PTX-PM (10%) in a syngeneic 4T1-luc breast tumor model based on measurements of tumor volume, 4T1-luc breast tumor bioluminescence, and survival. Importantly, intravenous administration of o(LA)₈-PTX-PM is well tolerated by BALB/c mice. In summary, oligo(lactic acid)₈-PTX is more compatible than PTX with PEG-b-PLA micelles, more stable, and may expand the role of PEG-b-PLA micelles from "solubilizer" into "nanocarrier" for PTX as a next-generation taxane for cancer.

Keywords: Block copolymer; Oligo(lactic acid); PEG; Polymeric micelle; Prodrug; Taxanes.

Figure 1.

An o(LA)₈-PTX prodrug-loaded PEG-b-PLA micelle (o(LA)₈-PTX-PM). Relative to a PTX-loaded PEG-b-PLA micelle (PTX-PM), o(LA)₈-PTX-PM may be more stable in blood. After release, o(LA)₈-PTX converts into PTX, lactic acid-PTX, and o(LA)₂-PTX (o(LA)_0–2-PTX) as metabolites for a lower C_max and higher plasma AUC relative to PTX-PM, aiming for reduced toxicity and higher antitumor efficacy.

Figure 2.

(A) Images of PTX-PM (10%), o(LA)₈-PTX-PM (10%), and o(LA)₈-PTX-PM (50%) at 2 mg/mL drug level at 25 °C after 24 hours, (B) Particle size analysis by dynamic light scattering (DLS) of PTX-PM (10%), o(LA)₈-PTX-PM (10%), and o(LA)₈-PTX-PM (50%), (C) Representative AFM image of o(LA)₈-PTX-PM (50%), and (D) In vitro drug release profiles of PTX from PTX-PM (10%) (drug precipitation < 4 hours), o(LA)₈-PTX from o(LA)₈-PTX-PM (10%), and o(LA)₈-PTX-PM (50%) in 10 mM PBS at 37 °C and pH 7.4 (mean ± SD, n=3).

Figure 3.

In vitro rat plasma stability of (A) o(LA)₈-PTX, (B) o(LA)₈-PTX-PM (10%), and (C) o(LA)₈-PTX-PM (50%) at a final concentration of 10 μM at 37 °C (mean ± SEM, n=3).

Figure 4.

In vitro cytotoxicity of PTX, o(LA)₂-PTX, o(LA)₈-PTX, PTX-PM (10%), o(LA)₈-PTX-PM (10%), and o(LA)₈-PTX-PM (50%) for 4T1-luc cells (mean ± SEM, n=4) (**. p < 0.01; *, p < 0.05).

Figure 5.

(A) Concentration-time profile in rat plasma and (B) area under the concentration-time curve (AUC_0–24) of PTX from PTX-PM (10%) at 10 mg/kg, o(LA)_0–2-PTX and o(LA)_0–8-PTX from o(LA)₈-PTX-PM (10%) at 10 mg/kg PTX-eq and o(LA)_0–2-PTX and o(LA)_0–8-PTX from o(LA)₈-PTX-PM (50%) at 10 mg/kg PTX-eq in male Sprague-Dawley rats. (mean ± SEM, n=4; ***, p < 0.001; **, p < 0.01; *, p < 0.05). Relative intensity of o(LA)_0–8-PTX over time in rat plasma after IV injection of (C) o(LA)₈-PTX-PM (10%) at 10 mg/kg PTX-eq, and (D) o(LA)₈-PTX-PM (50%) at 10 mg/kg PTX-eq in male Sprague-Dawley rats (Digits in Figures 5C and 5D represent the number of lactic acid oligomers (n) of o(LA)_n-PTX; mean ± SEM, n=4).

Figure 6.

In vivo antitumor efficacy in BALB/c mice bearing s.c. 4T1-luc breast tumors. Treatments were initiated 14 days post tumor implantation: PTX-PM (10%), o(LA)₈-PTX-PM (10%), and o(LA)₈-PTX-PM (50%) IV injection at 60 mg/kg PTX-eq (qwx3), indicated by arrows. (A) Whole-body bioluminescence images of 4T1-luc mice, (B) Relative tumor volume (mean ± SEM, n=3–4; *: p < 0.05 (o(LA)₈-PTX-PM (50%) vs. o(LA)₈-PTX-PM (10%), PTX-PM (10%), or saline control), (C) Relative body weight change, (D) % bioluminescence intensity of region of interest (ROI) (mean ± SEM, n=3; *: p < 0.05 (o(LA)₈-PTX-PM (50%) vs. o(LA)₈-PTX-PM (10%), PTX-PM (10%), or saline control), and (E) Kaplan-Meier analysis for survival (> 1200 mm³ endpoint tumor volume; **: p < 0.01 (o(LA)₈-PTX-PM (50%) vs. PTX-PM (10%)).

Figure 7.

(A) Relative body weight of female BALB/c mice over time after single tail-vein injection: PTX-PM (10%) at 100 mg/kg, o(LA)₈-PTX-PM (10%) at 100 mg/kg PTX-eq, and o(LA)₈-PTX-PM (50%) at 100, 150, and 200 mg/kg PTX-eq, respectively (mean ± SEM, n=3). Two mice suffered from loss of consciousness, flushed skin, and dyspnea were euthanized on day 0 and day 1 for PTX-PM (10%) at 100 mg/kg. (B) Relative body weight of female BALB/c mice over time after daily injection on days 0, 1 and 2 of PTX-PM (10%) at 50 mg/kg, o(LA)₈-PTX-PM (10%) at 50 mg/kg PTX-eq, and o(LA)₈-PTX-PM (50%) at 50 mg/kg PTX-eq (mean ± SEM, n=5; *: p < 0.05 (o(LA)₈-PTX-PM (50%) vs. PTX-PM (10%)); **: p < 0.01 (saline vs. PTX-PM (10%)).