Targeted drug delivery by novel polymer-drug conjugates containing linkers cleavable by disease-associated enzymes

Abstract

We have conceptualized a new class of polymer-linker-drug conjugates to achieve targeted drug delivery for the systemic treatment of cancer and other inflammatory diseases. The physiochemical properties of the polymer allow the conjugate to circulate longer in the body by minimizing renal and hepatic clearance, thereby improving the pharmacokinetics of the attached drugs. Traditionally, linkers are degraded by acidity or by some ubiquitous intracellular enzymes. We incorporate linkers that are sensitive to a specific extracellular enzyme whose overexpression is co-localized with the diseased tissue. The drug molecules remain inactive when attached to the polymer, thus preventing normal tissues from harmful side effects. When the conjugate is transported to the diseased area where there is a high level of the target enzyme, the linkers are cleaved to release the drugs at the specific site. As an example, we designed and synthesized two generations of novel polymer-peptide-drug conjugates for the tumor-targeted delivery of chemotherapeutics. To allow for passive targeting and enhanced permeation and retention (EPR), dextran with a size greater than 6 nm was selected as the polymeric carrier. This biocompatible and biodegradable carrier was chemically modified to allow for conjugation with doxorubicin and methotrexate, two common chemotherapeutics with undesirable side effects.

(cont.) Since matrix-metalloproteinases (MMPs) are associated with a number of types of cancer and their functions are essential to disease progression, including degrading extracellular matrix, releasing angiogenic factors and activating growth factors, we explored the possibility of MMP-mediated drug release. The synthesis procedures combined solid phase and solution phase techniques to enable flexibility in the linker design and in the charge modification of the polymer. This scaleable and robust process produced new conjugates that demonstrated excellent stability under physiological conditions and optimized sensitivity to enzymatic cleavage by MMP-2 and MMP-9. The new conjugate, dextran-peptide-methotrexate, was assessed for its in vivo anti-tumor efficacy and systemic side effects. It was compared to free methotrexate and a similar conjugate, differing by an MMP-insensitive linker, at equivalent intraperitoneal dosages administered weekly. The MMP-sensitive conjugate resulted in effective inhibition of in vivo tumor growth in each of the two separate tumor models that overexpress MMP-2 and MMP-9 (HT-1080 and U- 87). In contrast, free methotrexate resulted in no significant tumor reduction in the same models. Neither free methotrexate nor the conjugate caused any tumor inhibition in mice bearing RT- 112, a slower-growing model which expresses significantly less MMP than HT-1080 and U-87 . The anti-proliferative effect of the drug contributed to the inhibition of tumor growth. Systemic side effects caused by the MMP-sensitive conjugates were tolerable.

(cont.) MMP-insensitive conjugates, though able to inhibit tumor growth, caused toxicity in the small intestine and bone marrow and the experiment was terminated after one injection. We conducted a biodistribution study in HT-1080 bearing mice to investigate the targeting mechanism of the new conjugate. Independent of the linker sequence, passive targeting was evidenced by the prolonged plasma circulation and higher tissue accumulations of the conjugates in comparison with free methotrexate. The ratios of drug accumulation at the tumor versus the major site of side effects (small intestine) for both conjugates were enhanced by the EPR effects. The difference in the drug accumulation at the tumor site was insignificant between conjugates with MMP-sensitive and MMP-insensitive linkers. We concluded that the tumor targeting effect of the dextran-peptide-methotrexate conjugate was dominantly due to passive targeting and EPR. The difference in the systemic side effects observed for the conjugates with different linkers was attributed to their varying susceptibility towards enzymes in normal tissues.