Intra-Lesional Injection of the Novel PKC Activator EBC-46 Rapidly Ablates Tumors in Mouse Models

Intra-lesional chemotherapy for treatment of cutaneous malignancies has been used for many decades, allowing higher local drug concentrations and less toxicity than systemic agents. Here we describe a novel diterpene ester, EBC-46, and provide preclinical data supporting its use as an intra-lesional treatment. A single injection of EBC-46 caused rapid inflammation and influx of blood, followed by eschar formation and rapid tumor ablation in a range of syngeneic and xenograft models. EBC-46 induced oxidative burst from purified human polymorphonuclear cells, which was prevented by the Protein Kinase C inhibitor bisindolylmaleimide-1. EBC-46 activated a more specific subset of PKC isoforms (PKC-βI, -βII, -α and -γ) compared to the structurally related phorbol 12-myristate 13-acetate (PMA). Although EBC-46 showed threefold less potency for inhibiting cell growth than PMA in vitro, it was more effective for cure of tumors in vivo. No viable tumor cells were evident four hours after injection by ex vivo culture. Pharmacokinetic profiles from treated mice indicated that EBC-46 was retained preferentially within the tumor, and resulted in significantly greater local responses (erythema, oedema) following intra-lesional injection compared with injection into normal skin. The efficacy of EBC-46 was reduced by co-injection with bisindolylmaleimide-1. Loss of vascular integrity following treatment was demonstrated by an increased permeability of endothelial cell monolayers in vitro and by CD31 immunostaining of treated tumors in vivo. Our results demonstrate that a single intra-lesional injection of EBC-46 causes PKC-dependent hemorrhagic necrosis, rapid tumor cell death and ultimate cure of solid tumors in pre-clinical models of cancer.

病灶内化疗（Intra-lesional chemotherapy）用于治疗皮肤恶性肿瘤（cutaneous malignancies）已有数十年历史，相较全身给药制剂，其可实现更高的局部药物浓度且毒性更低。本文报道了一种新型二萜酯（diterpene ester）EBC-46，并提供了支持其作为病灶内治疗手段的临床前（preclinical）数据。
在一系列同基因（syngeneic）模型与异种移植（xenograft）模型中，单次注射EBC-46可引发快速炎症反应与血液涌入，随后形成焦痂并快速实现肿瘤消融（tumor ablation）。EBC-46可诱导纯化的人多形核细胞（purified human polymorphonuclear cells）发生氧化爆发（oxidative burst），该效应可被蛋白激酶C（Protein Kinase C, PKC）抑制剂双吲哚马来酰亚胺-1（bisindolylmaleimide-1）阻断。相较于结构相关的佛波醇12-肉豆蔻酸酯13-乙酸酯（phorbol 12-myristate 13-acetate, PMA），EBC-46可激活更具特异性的PKC同工型（PKC isoforms）亚群，即PKC-βI、-βII、-α与-γ。
尽管体外（in vitro）实验显示EBC-46抑制细胞生长的效力较PMA低三倍，但体内（in vivo）实验中其治愈肿瘤的效果更为优异。注射后4小时通过离体（ex vivo）培养未检测到存活肿瘤细胞。对给药小鼠的药代动力学（pharmacokinetic）特征分析显示，EBC-46优先滞留在肿瘤组织内；相较于注射至正常皮肤，病灶内注射后可引发更显著的局部反应，包括红斑（erythema）与水肿（oedema）。
联合注射双吲哚马来酰亚胺-1可降低EBC-46的治疗效力。通过体外内皮细胞单层（endothelial cell monolayers）通透性升高以及体内给药肿瘤的CD31免疫染色（CD31 immunostaining），证实了治疗后血管完整性（vascular integrity）的丧失。
本研究结果表明，在癌症临床前模型中，单次病灶内注射EBC-46可引发依赖于PKC的出血性坏死（hemorrhagic necrosis）、快速肿瘤细胞死亡，并最终实现实体瘤（solid tumors）的治愈。