Syntheses of the novel polymer-bound platinum-based chemotherapeutic agent poly(HPMA)-GGG-Ama=Pt=(1R,2R)-DACH, AP5346, and its precursors are reported. The method utilized in preclinical development of AP5346 is described herein. Additionally, an improved synthesis, which has shown that ion exchange resins can be removed, significantly less platinum can be used when the reaction mixture is pH-stated, and the synthesis can be performed at a higher concentration, is reported. These combined improvements result in a more cost-effective, scaleable procedure. Various methods of analysis of the drug substance are also discussed. Specifically, (1)H NMR spectroscopy is used for identity and can also distinguish small molecule impurities to below 0.1%. (195)Pt NMR determines the coordination environment of the platinum and also identity and purity in relation to platinum chelation of the construct. Size exclusion chromatography is used to establish the molecular weight of AP5346 while ICP-AES determines platinum content and platinum release rates in phosphate-buffered saline. The cumulative results of this work have yielded an efficient syntheses of a polymer-based chemotherapeutic agent with subsequent detailed characterization methods.
Purpose: AP5346 is designed to target a diaminocyclohexane platinum (Pt) moiety to tumors through pH-sensitive linkage to a 25 kDa hydroxypropylmethacrylamide polymer. The goal of these studies was to determine the rate of release of Pt as a function of pH, the antitumor activity, and plasma and tumor pharmacokinetics of AP5346 in preclinical models. Experimental Design: Antitumor activity was assessed in mice bearing B16F10 melanoma and M5076 and 2008 ovarian carcinomas. Pt levels in plasma, tumors, and tumor DNA were measured by atomic absorption and inductively coupled plasma mass spectrometry. Results: AP5346 did not release Pt when suspended in 5% dextrose and released only 3.5% of its Pt in 24 hours in buffer at pH 7.4; the rate of release was 7-fold higher at pH 5.4. When given at their respective maximum tolerated doses, the antitumor activity of AP5346 was superior to that of oxaliplatin against both the B16 melanoma and 2008 human ovarian carcinoma. It was more effective than cisplatin in both cisplatin-sensitive and cisplatin-resistant variants of the M5076 tumor. When given at equitoxic doses, the peak plasma concentration was 25-fold higher, and AUC (0-1) was 93 times higher, for AP5346 than for oxaliplatin. AP5346 delivered 16.3-fold more Pt to the tumor and 14.2-fold more Pt to tumor DNA than oxaliplatin based on AUC . Conclusions: AP5346 has a substantially better therapeutic index than oxaliplatin. AP5346 produced a marked increase in the delivery of diaminocyclohexane Pt to the tumor and tumor DNA over and above that attainable with oxaliplatin.
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