Interaction of PtCl4(Fast Black)2 with hyperthermia.

We are developing complexes of negatively charged PtCl4 with positively charged nuclear dyes as new antitumor agents for use alone and in conjunction with hyperthermia and/or radiation. Elemental analysis has shown that the complex PtCl4(Fast Black)2 is a tight ion pair. In experimentally growing EMT6 cells in vitro, PtCl4(Fast Black)2 killed cells in a log-linear manner which increased as the temperature of the exposures was increased from 37 to 42 degrees C or 43 degrees C. In addition, cell kill was also increased under conditions of low pH (6.45), especially in hypoxic cells treated at elevated temperature. Measurement of intracellular platinum levels after exposure to 25 microM cisplatin or PtCl4(Fast Black)2 demonstrated that platinum levels were between 170- and 200-fold higher after exposure to PtCl4(Fast Black)2. In vivo studies in the FSaIIC murine fibrosarcoma showed, again, that PtCl4(Fast Black)2 killed in a log-linear manner. Treatment of tumors placed in the thigh with 43 degrees C, 30-min hyperthermia immediately following i.p. injection of PtCl4(Fast Black)2 was dose modifying. One hundred mg/kg of PtCl4(Fast Black)2 produced a 4.6-day tumor growth delay which increased to 6.4 days with 43 degrees C, 30-min hyperthermia immediately following i.p. injection of PtCl4(Fast Black)2 was does modifying. One hundred mg/kg of PtCl4(Fast Black)2 produced a 4.6-day tumor growth delay which increased to 6.4 days with 43 degrees C, 30-min hyperthermia (growth delay for hyperthermia alone was 1.4 days), and 500 mg/kg produced a 5.6-day delay which increased to 11.0 days with hyperthermia. In contrast, cisplatin (5 mg/kg) produced a 4.4-day delay which increased to 5.9 days with hyperthermia. PtCl4(Fast Black)2 was well tolerated by animals, and the maximally tolerated dose was approximately 650 mg/kg. This new complex appears quite active as an antitumor agent alone and in conjunction with hyperthermia, and, since other studies have shown it to interact positively with radiation, this agent seems a very appropriate candidate for further development as a clinical anticancer drug.

Evidence for enzymatic activation and oxygen involvement in cytotoxicity and antitumor activity of N,N',N''-triethylenethiophosphoramide.

The cytotoxicity of N,N',N''-triethylenethiophosphoramide (thiotepa) was studied in vitro in the MCF-7 human breast carcinoma cell line and in vivo using the EMT6 mouse mammary tumor model, under various conditions of oxygenation and in the presence and absence of Aroclor 1254-induced liver preparations. The cytotoxicity of thiotepa toward exponentially growing MCF-7 cells was markedly dependent on the presence of oxygen during the period of drug exposure, with 3 log greater cell kill at 500 microM thiotepa being observed when the cells were normally oxygenated compared with hypoxic cells. Incubation of thiotepa with an Aroclor 1254-induced rat liver S-9 homogenate, in the presence of a NADPH-regenerating system, resulted in an 8-fold increase in cytotoxicity towards the MCF-7 cells over a wide range of drug concentrations. Thiotepa was shown to be metabolized under these conditions in a NADPH- and O2-dependent reaction that was catalyzed by one or more microsomal cytochrome P-450 enzymes that were present in the S-9 fraction. The thiotepa metabolite triethylene phosphoramide, which hydrolyzes significantly faster than thiotepa, was significantly less cytotoxic toward the MCF-7 cells than was thiotepa itself, suggesting that it is unlikely to be the S-9 metabolite responsible for the observed increase in drug cytotoxicity. Moreover, triethylene phosphoramide cytotoxicity was only partially O2 dependent and was largely unaffected by incubation in the presence of the S-9 preparation, indicating a mechanism of action distinct from that of thiotepa. Tumor cell survival experiments with the EMT6 mouse mammary carcinoma system revealed that a 3.6-fold increase in thiotepa cytotoxicity was obtained by prior administration of the liver inducer Aroclor 1254 to the tumor-bearing animals, 5 days before drug treatment. Finally, the therapeutic effectiveness of thiotepa was significantly enhanced (3- to 5.8-fold increase in tumor growth delay) when an increase in oxygenation was achieved, by carbogen breathing, in animals given the perfluorochemical emulsion Fluosol-DA. These findings establish that the cytotoxic effects of thiotepa are oxygen dependent and may involve, at least in part, metabolic processes catalyzed by cytochrome P-450 enzymes.

Oxygenation of tumors by a hemoglobin solution.

Tumor oxygen tensions were measured using a computer-controlled PO2 microelectrode in two preclinical solid tumor models, the rat 9L gliosarcoma and the rat 13672 mammary carcinoma. Tumor oxygenation profiles were determined under four conditions: (a) during normal air breathing, (b) during carbogen breathing, (c) after intravenous administration of a solution of ultrapurified polymerized bovine hemoglobin with normal air breathing and (d) after intravenous administration of a solution of ultrapurified polymerized bovine hemoglobin with carbogen breathing. Both tumors had severely hypoxic regions under normal air-breathing conditions. Although carbogen breathing increased the oxygenation of the better-oxygenated portions of the tumor, it made no impact on the severely hypoxic tumor regions. Administration of the hemoglobin solution was effective in increasing the oxygenation throughout both tumors under normal air-breathing conditions. The addition of carbogen breathing to administration of the hemoglobin solution eliminated severe hypoxia in the 9L gliosarcoma and markedly reduced the severely hypoxic regions of the 13672 mammary carcinoma. At 24 h after administration of the hemoglobin solution the 13672 mammary carcinoma showed greater hypoxia than before treatment, which was partially corrected with carbogen breathing.

Interaction of platinum complexes of thiazin and xanthene dyes with hyperthermia.

In an attempt to develop platinum-containing drugs for use with hyperthermia that would be relatively nontoxic at 37 degrees C but would become very cytotoxic at 42 degrees or 43 degrees C, several nuclear dyes were complexed to the tetrachloroplatinum(II) dianion (PtCl4) at a ratio of 2:1. The cytotoxicity of PtCl4 complexes of three thiazin dyes (thionin, azure B, and methylene blue), the xanthene dye pyronin Y, and the thiazole dye thioflavin was examined in exponentially growing euoxic and hypoxic EMT6 cells in vitro at 37 degrees, 42 degrees, and 43 degrees C and at pH 7.40 and 6.45. Of the thiazin dye complexes, the cytotoxicity of Pt(methylene blue)2 was most enhanced at hyperthermic temperatures. Both Pt(pyronin Y)2 and Pt(thioflavin)2 also became markedly more cytotoxic at 42 degrees and 43 degrees C at pH 6.45 vs pH 7.40. In vivo tumor excision assays in the FSaIIC fibrosarcoma showed that with each of the thiazin dye-platinum complexes, hyperthermia enhanced cell kill [most effectively on Pt(methylene blue)2] but was not dose-modifying. For both Pt(pyronin Y)2 and Pt(thioflavin)2, however, administration of 43 degrees C, 30-min hyperthermia to the tumor immediately after i.p. drug injection was dose-modifying. Tumor growth delay studies in the FSaIIC tumor system demonstrated that, as with the in vitro studies, Pt(pyronin Y)2 and Pt(methylene blue)2 were most enhanced by hyperthermia [tumor growth delay increased by 4.8- and 3.0-fold, respectively, vs only 1.3-fold for cisplatin (CDDP)]. Examination of intracellular platinum levels after exposure of EMT6 cells to 25 microM of drug for 1 h at 37 degrees and 42 degrees C and at pH 7.40 and 6.45 showed that each platinum-dye complex achieved platinum levels that were 100-600 times higher at 37 degrees C and pH 7.40 than those obtained using CDDP. The platinum levels for each drug dropped markedly when exposure took place at pH 6.45. Exposure at 42 degrees C only moderately increased platinum levels in cells exposed to these drugs. Thus, for several of these drugs the level of cytotoxicity observed was in great part independent of the intracellular platinum levels achieved. Pt(pyronin Y)2 is an effective drug for use with hyperthermia, and further studies using this combination with and without radiation are under way.

Schedule dependent tumour growth delay, DNA cross-linking and pharmacokinetic parameters in target tissues with cis-diamminedichloroplatinum(II) and etanidazole with or without hyperthermia or radiation.

It has been reported previously that striking increases in tumour growth delay and cytotoxicity are seen when cis-diamminedichloroplatinum(II) (CDDP) is combined with mild local hyperthermia (43 degrees C, 30 min) and/or etanidazole (ETA). This paper reports a study of CDDP pharmacology and the in vivo tumour DNA cross-linking produced by these combinations. In C3H mice bearing the FSaIIC murine fibrosarcoma, Pt plasma pharmacokinetics were not significantly altered by any of the combination of treatments. Although ETA caused no significant change in CDDP tissue pharmacokinetics, treatment of the tumour-bearing limb with hyperthermia immediately following an i.p. injection of CDDP (10 mg/kg) resulted in an increased peak Pt concentration (3.5 versus 2.8 micrograms Pt/g tumour wet weight) and doubled the t1/2 elimination of Pt (15 to 30 h) from the tumour. Similar heat-induced changes were observed in the Pt pharmacokinetics in skin. There was about a three-fold increase in the Pt area under the curve (AUC) for the tumour, a 1.5-fold increase in the AUC for skin and little change in the AUC for muscle with hyperthermia. When the tumour DNA cross-linking factor (CLF) was determined, it was found that local hyperthermia treatment (43 degrees C, 30 min) increased the CLF of CDDP from 1.7 to 2.7 and hyperthermia (43 degrees C, 1 h) further increased the CLF to 6.1. Misonidazole (MISO) (1 g/kg) increased the CDDP CLF to 2.0, 6.3 and 15.1 in conjunction with 37, 43 (30 min) and 43 degrees C (1 h), respectively. ETA (1 g/kg) was more effective than MISO at increasing the CDDP CLF, producing CLFs of 2.8, 9.1 and 21.5 at 37, 43 (30 min) and 43 degrees C (1 h), respectively. These changes in CLF were reflected in an increased tumour growth delay in the FSaIIC murine fibrosarcoma with CDDP (5 mg/kg) alone from 4.4 to 5.9 days with 43 degrees C (30 min) and then to 11.9 days with ETA (1 g/kg) and 20.9 days with both ETA and local hyperthermia (43 degrees C, 30 min). When CDDP, ETA and hyperthermia were added to a radiation schedule of 300 cGy daily for five days, it was found that giving ETA (1 g/kg), CDDP (5 mg/kg) and hyperthermia (43 degrees C, 30 min) together on day 1 produced the largest tumour growth delay (43 days) and that other schedules which divided the dose of ETA over the other days of the radiation treatment (including one schedule with a second heat treatment on day 4) were significantly inferior.(ABSTRACT TRUNCATED AT 400 WORDS)