Systemic Chemotherapy of Melanoma: NMR Studies of Lonidamine & N-Mustard Activity

描述（由申请人提供）：黑色素瘤仍然是最致命的人类癌症之一，没有有效的方法来治疗这种播散性疾病。我们建议通过[肿瘤]选择性[细胞内]酸化使黑色素瘤对N-芥子烷化剂敏感来开发一种有效的黑色素瘤全身化疗方法。我们的策略是将葡萄糖与单羧酸转运（MCT）抑制剂氯尼达明LND共同给药，以捕获肿瘤中糖酵解产生的乳酸，并通过阻碍丙酮酸转运至线粒体来抑制氧化代谢。这种方法依赖于MCT是肿瘤细胞内pH（pHi）稳态的关键途径，这已在我们的黑色素瘤模型中得到证实。既往在高血糖条件下使用LND治疗导致了一定的死亡率。我们在该2年R 01上获得资助，以开发一种安全、可重现的方法，用于酸化含LND的人DB 1黑色素瘤异种移植物。通过腹膜内给予LND 100 mg/kg（不含葡萄糖），我们实现了肿瘤酸化至pHi为6.4±0.1，持续至少1小时，在此期间肿瘤的NTP/Pi比值降低50%;没有死亡，没有显著的体重减轻，并且与该治疗相关的毒性最小。[LND诱导正常组织的细胞外pH（pHe）的最小变化，但从pHi 6.9、pHe 7.0或！pH=~0至pHi=6.4、pHe=6.85或ΔpH=-0.45的处理后值。肿瘤中的稳态乳酸盐相对于LND前水平增加了3倍。呼吸、心电图、电解质水平和血氧没有变化，而血液分析仍在进行中。骨骼肌的pHi和ATP/Pi没有变化，肝脏的pHi和ATP/Pi只有短暂的小幅度下降。美法仑（LPAM 7.5 mg/kg i. v.）+ LND的生长延迟时间为10.5±0.5 d，而单独LND为0.6±0.7 d。LPAM单用组为1.4± 0.1d。LPAM对酸化的[增强的]响应归因于活性中间体（aziridinium离子）浓度的增加、由于谷胱甘肽-S-转移酶活性降低而导致的GSH降低以及由于O 6-烷基转移酶的酸抑制而导致的DNA修复降低。肿瘤体积减少52.5 ± 12.5%，与估计的log 10细胞杀伤=0.3012 = log 10（2.0）一致。这些初步研究为实施人黑色素瘤异种移植模型的多剂量全身治疗奠定了基础，为最终的临床试验做准备。该更新提案的目的是：1）通过低水平葡萄糖与LND共同给药，最大化肿瘤酸化，同时仍避免危及生命的毒性，并将这些研究扩展到糖酵解作用更强的DB 8黑色素瘤人异种移植模型。2)由于其他N-芥子类药物在酸性pHi值下可能表现出类似的活性增强，但毒性可能较低，因此将在单剂量和多剂量给药下将LPAM与环磷酰胺、苯丁酸氮芥和苯达莫司汀进行比较。3)评价可能用于无创早期检测反应的1H NMR标记物-MRS测定的乳酸和总胆碱以及MRI测定的表观扩散常数（ADC）和T2。

DESCRIPTION (provided by applicant): Melanoma remains one of the deadliest of human cancers with no effective method for treating the disseminated disease. We propose to develop an effective method for systemic chemotherapy of melanoma by [tumor] selective [intracellular] acidification to sensitize melanomas to N-mustard alkylating agents. Our strategy is to coadminister glucose with the monocarboxylic acid transport (MCT) inhibitor lonidamine LND in order to trap lactic acid produced by glycolysis in the tumor and to inhibit oxidative metabolism by impeding pyruvate trans- port into mitochondria. This approach depends on the MCT being the key pathway for tumor intracellular pH (pHi) homeostasis, which has been confirmed in our melanoma models. Previous treatment with LND under hyperglycemic conditions led to some mortality. We were funded on this 2yr R01 to develop a safe reproducible method for acidification of human DB1 melanoma xenografts with LND. By administering LND 100 mg/kg i.p. without glucose, we have achieved acidification of the tumor to a pHi of 6.4±0.1 for at least hr during which time the NTP/Pi ratio of the tumor decreased by 50%; there was no mortality, no significant loss of weight and minimal toxicity associated with this treatment. [LND induced minimal changes in extracellular pH (pHe) of normal tissue, but modified the pH gradient across the plasma membrane of the tumor from pretreatment values of pHi 6.9, pHe 7.0 or !pH=~0 to a post-treatment values of pHi=6.4, pHe=6.85 or ΔpH=-0.45. Steady state lactate in the tumor increased 3-fold relative to pre-LND levels.] Respiration, EKG, electrolyte levels and blood oxygenation did not change, while blood analysis is still in progress. The pHi and ATP/Pi of skeletal muscle did not change, and there was only a small transient decrease in pHi and ATP/Pi of the liver. Melphalan (LPAM 7.5 mg/kg i.v.)+ LND exhibited a growth delay of 10.5±0.5 d vs. LND alone, 0.6±0.7 d. and LPAM alone 1.4±0.1 d. The [enhanced] response of LPAM to acidification is attributed to an increase in the concentration of the active intermediate (aziridinium ion), a decrease in GSH due to decreased activity of glutathione-S-transferase and a decrease in DNA repair resulting from acid inhibition of O6-alkyl-transferase. Tumor volume decreased by 52.5 ±12.5%, consistent with the estimated log10 cell kill=0.3012 = log10(2.0). These preliminary studies set the stage for implementation of multidose systemic treatment of xenograft models of human melanoma in preparation for eventual clinical trials. The Aims of this renewal proposal are: 1) To maximize tumor acidification while still avoiding life threatening toxicity by coadministration of low levels of glucose with LND, and to also extend these studies to the more glycolytic DB8 melanoma human xenograft model. 2) Since other N-mustard agents might exhibit similar enhancements of activity at acidic pHi values but perhaps with lower toxicity, LPAM will be compared with cyclophosphamide, chlorambucil and bendamustine under single and multidose administration. 3) To evaluate potential 1H NMR markers for noninvasive early detection of response--lactate and total choline by MRS and apparent diffusion constant (ADC) and T2 measured by MRI.