Bioreductive Agents Research Articles

In vitro cytotoxicity and chemosensitizing activity of the dual function nitroimidazole RB 6145

Purpose : To determine the cytotoxicity and chemosensitizing potential of RB 6145 in mouse KHT/iv and human A549 tumor cells. Methods and Materials : RSU 1069, the lead compound in a series of nitroimidazoles containing an alkylating aziridine function, has been shown to possess a high degree of selective cytotoxicity for hypoxic cells in addition to being a potent sensitizer of radiation and chemotherapy. Unfortunately, preliminary clinical studies have revealed a dose-limiting gastrointestinal toxicity for RSU 1069. Recently RB 6145, the ring-opened analogue of RSU 1069, has been found to be less emetic than RSU 1069. In the present studies, we assessed both the differential hypoxic cell cytotoxicity of RB 6145 and its chemosensitizing potential when combined concomitantly with variable doses of the activated form of cyclophosphamide (4-hydroperoxy-cyclophosphamide, 4-OOH-CP) or the nitrosourea CCNU. Results : As we had observed previously for RSU 1069, RB 6145 was found to be less cytotoxic to human than rodent tumor cells. In addition, the degree of selective cytotoxicity toward hypoxic cells was (a) less in A549 than in KHT/iv cells (factor of 9 vs. 80) but (b) comparable to that seen with RSU 1069. For both cell lines, inclusion of the sensitizer enhanced the cell killing of the chemotherapeutic agent 4-OOH-CP by a factor of ∼ 1.5–1.7-fold. When combined with CCNU, RB 6145 increased the killing of A549 cells ∼ 1.8-fold. Similar hypoxic cell preferential cytotoxicity and enhancement in anti-tumor treatment efficacy were seen when A549 cells were exposed to the R enantiomer of RB 6145 (PD 144872) either alone or in combination with CCNU. Conclusion : These data support the notion that further consideration should be given to the clinical application of these Bioreductive agents.

Peri-tumoural hypoxia in human brain: peroperative measurement of the tissue oxygen tension around malignant brain tumours.

Malignant brain tumours contain focal hypoxic areas that may increase their resistance to chemotherapy and radiotherapy. Following surgical excision, the peri-tumoural area will contain residual viable tumour cells, and this area is therefore the logical site for subsequent therapy. The new bioreductive agents are metabolized under hypoxic conditions to produce a cytotoxic species. Peroperative peri-tumoural micro-polarographic measurements have been made to establish the oxygen environment of this region and to determine whether the hypoxic conditions might allow for bioreductive drug activation. The micro-polarographic method is described and results are presented for "normal" white matter (8 patients) to allow comparison with peri-tumoural brain (8 patients) before and after removal of the tumour. The results suggest that peri-tumoural brain (median pO2 10.8 mmHg, 18% pO2 < 2.5 mmHg) is markedly hypoxic in comparison with the "normal" brain (median pO2 15.3 mmHg, less than 2% < 2.5 mmHg), and that surgery improves peri-tumoural oxygenation towards that of the "normal" white matter. It is concluded that the hypoxic peri-tumoural area can provide the conditions under which bioreductive agents may be activated.

Hypoxia-selective antitumor agents derived from 1,9-diazaanthracene

The nitroacridine derivative 9-[3-( N,N-dimethylamino)propylamino]-1-nitroacridine (nitracrine) is a potent hypoxia-selective cytotoxin for tumor cells in culture. Modifications of the acridine ring result in altered DNA binding properties. This has suggested the possibility of preparing new nitracrine analogues retaining the nitro group and the alkylamino lateral chain. In this paper we describe the synthesis of the new diazaanthracenes 4, 5, 6 in order to study the relationships between the heterocyclic system and the hypoxia-selective cytotoxicity. This class of new bioreductive agents may constitute a group of potential interest for the design of new cytotoxins.

One-electron oxidation and reduction reactions of vitamin C derivatives: 6-bromo- and 6-chloro-6-deoxy-ascorbic acid.

6-Bromo- and 6-chloro-6-deoxy derivatives of ascorbate anion are able to transfer an electron to the oxidizing radicals .OH, Br2.- and RS. with the same rate constants as the ascorbate anion itself. The resulting radicals also show the same kinetic stabilities and optical absorption spectra as the well-characterized ascorbate radical anion (lambda max = 360 nm; epsilon 360 = 330 m2 mol-1). This proves that there is no influence of the structural changes in the side chain on the antioxidant capacity of the ascorbate moiety. In contrast, measured reduction of the 6-halo-6-deoxy derivatives occurs significantly faster (up to one order of magnitude) than the reduction of unsubstituted ascorbate. For example, absolute rate constants of 4.6 x 10(9) and 2.2 x 10(7) dm3 mol-1 s-1 have been measured for the reactions of bromo-derivative with eaq- and (CH3)2COH respectively. These radical-induced reductions proceed via dissociative electron capture and, under cleavage of the C-halogen bond, yield C-6 carbon-centered radicals. In the presence of oxygen the corresponding peroxyl radical is readily formed. This radical is again able to oxidize the ascorbate moiety (rate constant 2 x 10(7) dm3 mol-1 s-1). Results are discussed in terms of biological relevance of the investigated compounds regarding their ability to act as efficient antioxidants and bioreductive antitumour agents simultaneously.

Comparison of the enhancement of tumor responses to fractionated irradiation by SR 4233 (Tirapazamine) and by nicotinamide with carbogen

Purpose : This study was undertaken to compare in a fractionated regimen, with clinically relevant radiation doses, two radiation response modifiers that function by different mechanisms: SR 4233, a bioreductive agent toxic to hypoxic cells, and nicotinamide with carbogen, a combination that has been shown to improve tumor oxygenation. Methods and Materials : Cell survival assays were used to examine the response of three different tumors: KHT, RIF-1 and SCCVII/St in C3H/Km mice. Regrowth delay studies were also performed with the RIF-1 tumor. A fractionated irradiation schedule, consisting of twice daily 2.5 Gy treatments was investigated with and without drug pretreatment. SR 4233 was given IP at 0.12 mmol/kg one half hour before each irradiation. Nicotinamide (250, 500, 1000 mg/kg) was given IP 1 h before each irradiation with carbogen exposure 5 min prior to and during the irradiation. Results : Both treatment strategies enhanced the response of all three tumors to the fractionated radiation regimen. However, for two of the tumors (KHT and SCCVII), SR 4233 produced a significantly greater enhancement than did the combination of nicotinamide + carbogen. For the RIF-1 tumor (which has the lowest hypoxic fraction of the three), the response was comparable for the two modalities. For nicotinamide + carbogen, there was no significant change in the radiation enhancement at nicotinamide doses between 250 and 1000 mg/kg. Conclusion : Adding the bioreductive cytotoxin SR 4233 or nicotinamide + carbogen to fractionated irradiation enhances the response of the three transplanted tumors used in this study to fractionated irradiation. The radiation enhancement was significantly greater, however, for SR 4233 for two of the tumors with comparable results in the third. The data are consistent with the prediction that killing tumor hypoxic cells can produce a similar or greater enhancement of the efficacy of fractionated radiation in enhancing tumor response than either oxygenating or radiosensitizing these cells.

Effect of misonidazole and low‐oxygen treatment on mouse tumors

AbstractThe tumor cytotoxic activity of a bioreductive agent is dependent on the extent of tumor hypoxia. It is to be expected, therefore, that the tumor cytotoxicity of misonidazole will vary depending on whether it was administered to mice exposed only to an air‐breathing environment or whether it was administered either before mice were treated with 8% oxygen for 48 hr (maximal tumor levels of the drug will then coincide with an increase in tumor hypoxia) or after mice completed a 48‐hr 8% oxygen treatment (maximal tumor levels of the drug will now coincide with a reduction in the tumor hypoxic fraction). This was investigated using the 3‐methylcholanthrene‐induced rhabdomyosarcoma in BALB/c mice, the Fib/T tumor in WHT mice, and the CaNT tumor in CBA mice. The response of all three tumor types to the various treatments was determined by assessing post‐treatment tumor growth. An excision and in vitro colony‐forming assay was also used to assess Fib/T tumor response. Results indicated that only the 3‐methylcholanthrene‐induced rhabdomyosarcoma responded to misonidazole and only when the 48‐hr 8% oxygen exposure preceded drug administation. In explaining this result, it is proposed that misonidazole increases reperfusion damage that occurs when animals are returned to an air‐breathing environment following a low‐oxygen exposure. © Wiley‐Liss, Inc.

Activation of potassium-channels as a sensitive assay for oxidative stress

Drugs with two reducible centers, both of which must be metabolized by oxygen-inhibitable processes for full activation (“bis-bioreductive agents”), offer potential for the development of hypoxia-selective cytotoxins with improved oxygen sensitivity. The sidechain N-oxide (1-NCO) of the (mono)bioreductive agent nitracrine (1-NC) has been synthesized and evaluated as a potential example of such an approach. The association constant for reversible DNA binding of l-NCO was 15-fold lower than that of 1-NC, as measured by equilibrium dialysis in a low ionic strength buffer, indicating that the N-oxide has the potential to act as a less toxic pro-drug of 1-NC. Cell uptake and aerobic cytotoxicity of 1-NCO were much lower than for 1-NC whereas its hypoxic selectivity as a cytotoxin was greatly increased. In stirred suspension cultures of AA8 cells, pure (< 0.02% 1-NC) 1-NCO was 1000–1500 times more potent under hypoxia than in 20% 02. For 1-NC the corresponding ratio was 10 ± 1. 1-NCO had greater hypoxic selectivity in this system than misonidazole (ratio 11), RSU 1069 (ratio 25), 8Me5NQ (ratio 60), or SR 4233 (ratio 80). Studies of 1-NCO metabolism indicate rapid, OZ-inhibited reduction to INC. The data are consistent with a two-step bioactivation mechanism, with reduction of the N-oxide generating a DNA intercalator of increased binding affinity, followed by reduction of the nitro group of this DNA-targeted cytotoxin to form reactive cytotoxic metabolites.

Rationale for the use of aliphatic N-oxides of cytotoxic anthraquinones as prodrug DNA binding agents: a new class of bioreductive agent.

NAD(P)H dependent cytochrome P450's and other haemoproteins under hypoxia, mediate two-electron reduction of a wide range of structurally dissimilar N-oxides to their respective tertiary amines. Metabolic reduction can be utilised, in acute and chronic hypoxia, to convert N-oxides of DNA affinic agents to potent and persistent cytotoxins. In this respect a knowledge of N-oxide bioreduction and the importance of the cationic nature of agents that bind to DNA by intercalation can be combined to rationalise N-oxides as prodrugs of DNA binding agents. The concept is illustrated using the alkylaminoanthraquinones which are a group of cytotoxic agents with DNA binding affinity that is dependent on the cationic nature of these compounds. The actions of the alkylaminoanthraquinones involve drug intercalation into DNA (and double stranded RNA) and inhibition of both DNA and RNA polymerases and topoisomerase Type I and II. A di-N-oxide analogue of mitoxantrone, 1,4-bis([2-(dimethylamino-N-oxide)ethyl]amino)5,8-dihydroxyanthracene -9,10- dione (AQ4N) has been shown to possess no intrinsic binding affinity for DNA and has low toxicity. Yet in the absence of air AQ4N can be reduced in vitro to a DNA affinic agent with up to 1000-fold increase in cytotoxic potency. Importantly the reduction product, AQ4, is stable under oxic conditions. Studies in vivo indicate that antitumour activity of AQ4N is manifest under conditions that promote transient hypoxia and/or diminish the oxic tumour fraction. The advantage of utilising the reductive environment of hypoxic tumours to reduce N-oxides is that, unlike conventional bioreductive agents, the resulting products will remain active even if the hypoxia that led to bioactivation is transient or the active compounds, once formed, diffuse away from the hypoxic tumour regions. Furthermore, the DNA affinic nature of the active compounds should ensure their localisation in tumour tissue.

Effect of glucose-mediated pH reduction and cyclophosphamide on oxygenation of transplanted rat tumors

Purpose: Glucose-mediated reduction of tumor pH is under investigation as a means to improve the therapeutic index of anticancer agents. An improvement of glucose supply to tumors is, however, likely to influence various metabolic and pathophysiological parameters apart from pH which, in turn, could modulate H + ion-mediated effects. As a first step to identify changes in these parameters, we have investigated the effect of glucose-mediated pH reduction on oxygenation of malignant tissues either per se or in combination with a pH-sensitive drug, cyclophosphamide. Methods and Materials : H + ion and oxygen-sensitive semi-microelectrodes were used to measure pH and pO 2 in transplanted TVIA and AH13r rat tumors at normoglycemia and following high-dose intravenous glucose infusion. Results : In both tumors analyzed, pH reduction was accompanied by a decrease in pO 2. In TV1A tumors, for example, the mean (median) p0 2 decreased from 8.2 mm Hg (3.7 mm Hg) to 3.9 mm Hg (1.7 mm Hg) at 2 hr and 2.9 mm Hg (1.9 mm Hg) at 24 hr, respectively, after raising the plasma glucose concentration to 25 ± 2 mmol/l. At the same time points, the mean pH had declined from 6.89 to 6.29 and 6.24, respectively. The class of p0 2 readings < 5 mm Hg increased from a pretreatment value of 65% to ∼90% at 24 hr. In contrast, cyclophosphamide treatment resulted in improved oxygenation of AH13r tumors, an effect first observed at 24 hr after drug administration. When both modalities were combined, cyclophosphamide partly prevented the acidosis-associated decrease in oxygen partial pressure. Conclusion : The results of this study indicate that, within the context of the therapeutic approach used, cytotoxic agents should be employed which are more active against hypoxic than against well-oxygenated cells. In particular, glucose-mediated reduction of oxygen partial pressure in malignant tissues may be exploited to increase the fraction of hypoxic cells prior to administration of drugs activated in hypoxic cells by reductive metabolism (bioreductive agents).

In vitro activity of the benzotriazene dioxide SR 4233 against human tumour colony-forming units

SR 4233 (3-amino-1,2,4-benzotriazine 1,4-dioxide) is a novel bioreductive agent selectively toxic to hypoxic cells. It is active as a radiation sensitiser in vitro. Using a human tumour cloning system we have studied the effects of SR 4233 against freshly explanted human tumour specimens under hypoxic and non-hypoxic culture conditions. For hypoxic conditions, final concentrations of SR 4233 of 10.0–500 μmol/l were used in short-term (1 h) exposure experiments. Final concentrations in non-hypoxic experiments ranged from 10 to 1350 μmol/l. 25 tumour specimens were tested under each culture condition. Of those, 14 (56%) were evaluable. The most common tumour types recruited included ovarian, non-small cell lung, and breast cancer. A moderate concentration-dependent increase in the frequency of inhibited tumour specimens under non-hypoxic conditions was observed with zero out of 10 sensitive specimens at 10 μmol/l as compared with five out of 14 (36%) sensitive specimens at 500 μmol/l ( P < 0.02). However, when hypoxic conditions were used SR 4233 had a profound antitumour activity, (two out of 14 specimens sensitive at 10 μmol/l compared with 10 out of 10 specimens sensitive at 500 μmol/l, P < 0.00005). We conclude that SR 4233 is active against tumour colony-forming units in vitro and that its antitumour activity is greatly increased against hypoxic tumour cells.

Nitroheterocyclic-linked acridines as DNA-targeting bioreductive agents

Nitroheterocyclic-linked acridines as DNA-targeting bioreductive agents

Potentiation of antineoplastic drugs in vitro and in vivo by DNA intercalating bioreductive agents

AbstractThree recently synthesized 2‐nitroimidazole‐linked acridine DNA intercalators (NLA‐1, NLA‐2, and NLA‐4) have been studied as potentiators of the cytotoxic effect of melphalan (L‐PAM), 1‐(2‐chloroethyl)‐3‐cyclohexyl‐1‐nitrosourea (CCNU), cis‐diamminedichloroplatinum (II) (cis‐DDP), and doxorubicin in V79 cells. In vivo studies have also been performed with all three sensitizers and L‐PAM, while NLA‐1 alone was examined for potentiation of cis‐DDP, using C3H/HEJ mice, bearing a murine ovarian tumor. Significant chemosensitization was observed in vitro with L‐PAM, CCNU, and cis‐DDP after hypoxic preincubation treatment of the cells (37°C) at negligible concentrations of each sensitizer, while inhibition was observed with doxorubicin. Long hypoxic preexposure times at relatively low sensitizer doses were more effective than short hypoxic preexposure times at high sensitizer doses for potentiation. Glutathione depletion studies in vitro with diethyl maleate (DEM) showed that only less than half of the enhanced toxicity observed with NLA‐1 or NLA‐4 and L‐PAM is due to glutathione depletion by the sensitizer. The LD50/35 value in C3H/HEJ mice for NLA‐1, NLA‐2, and NLA‐4 was 45, 25, and 63 mg/kg, respectively. Significant antitumor effect was observed in vivo when 25 mg/kg of NLA‐1 was administered i.p. to tumor‐bearing mice 2 hr before L‐PAM administration (2.5 mg/ kg, i.p.). With this combination pattern of sensitizer:antineoplastic drug, 50% cures were observed in mice followed for &gt;150 days; no long‐term survivors were observed with L‐PAM therapy alone. © 1993 Wiley‐Liss, Inc.

Enhancement of the cytotoxicity of SR 4233 to normal and malignant tissues by hypoxic breathing.

The bioreductive cytotoxic agent SR 4233 (1,2,4-benzotriazine 3-amine 1,4-dioxide) has been shown to markedly potentiate the cell killing of mouse tumours when combined with fractionated radiation therapy. Differential metabolism under oxic compared to hypoxic conditions results in SR 4233 exhibiting selective cytotoxicity to hypoxic cells. This is thought to result from the production of a cytotoxic free radical which is generated predominantly in the absence of oxygen. We have examined a way of enhancing the effectiveness of this antitumour agent in vivo by artificially increasing the hypoxic fraction of tumours by hypoxic breathing. Mice are placed in a chamber containing 10% Oxygen 90% Nitrogen for 1 h after each administration of SR 4233. Our results in the SCCVII tumour model indicate that this manoeuvre results in a 10-fold increase in antitumour effectiveness of SR 4233 when administered in a fractionated regime with radiotherapy (8 x 2.5 Gy and 0.08 mmol kg-1), but not when a single treatment regime (1 x 20 Gy and 0.3 mmol kg-1) is used. Mathematical modelling of this effect is used to illustrate this phenomenon and can be used to predict the dependence of this type of therapy on the modification of tumour oxygenation.

Redox, Radiation, and Reductive Bioactivation

A brief review is presented of the background to, and the principles involved in, the development of redox-sensitive drugs for use in cancer therapy. The role of redox processes in the action of various types of radiosensitizers and in the activation of bioreductive drugs is described. The mechanisms by which many simple hypoxic cell radiosensitizers act are believed to involve fast electron transfer processes involving DNA. Some of these agents can also function as hypoxic cell cytotoxins, although the mechanisms involved are different. These "bioreductive drugs" are activated by intracellular metabolic reduction mediated through various cellular reductases. Usually, though not always, bioreduction is favored under hypoxic conditions, and this is why many of these compounds display differential cytotoxicity to hypoxic cells. This is one of the rationales for selectivity in solid tumors. The potencies of both hypoxic cell radiosensitizers and bioreductive drugs are strongly correlated with their electron affinities. Classes of bioreductive agents of current interest are described briefly. These include simple and dual-function nitroheterocycles including the highly potent compound RB-6145, quinone-based drugs including analogues of mitomycin C, and heterocyclic compounds containing N-oxide functions. The study of bioreductive agents for potential use as adjuncts for various approaches to cancer treatment is described.

Potentiation of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea's toxicity in vitro by two new bioreductive agents.

Two new bioreductive compounds, 9‐[3‐(2‐nitro‐1‐imidazolyl)propylamino]acridine hydrochloride (NLA‐1) and 9‐[2‐(2‐nitro‐1‐imidazolyl)ethylamino]acridine hydrochloride (NLA‐2), which behave as hypoxic cytotoxins and radiosensitizers, have been investigated for potentiation of 1‐(2‐chloroethyl)‐3‐cyclohexyl‐1‐nitrosourea's (CCNU) cytotoxic activity in vitro using V‐79 cells. The preincubation effect as well as conditions of coadministration of CCNU with each sensitizer have been examined. In this latter case, the median‐effect analysis was applied to evaluate whether the phenomenon was additive or synergistic. A clonogenic assay was used to score survival. Both bioreductive compounds, even at very low concentrations, significantly enhance the cytotoxic activity of CCNU under conditions of hypoxic preincubation. The enhancement of CCNU cytotoxicity is dependent upon preincubation time and the concentrations of both CCNU and the specific bioreductive agent. Coincubation of cells under hypoxia with CCNU and each bioreductive agent led to some potentiation, but only at lower survival levels. No chemosensitization was observed under aerobic conditions with either sensitizer.

Potentiation of alkylating chemotherapy by dual function nitrofurans in multi-cell spheroids and solid tumors

The cytotoxicity and chemosensitizing potential of four dual function nitrofurans was determined in human HT-29 multi-cell spheroids and rodent KHT sarcoma solid tumors. Spheroids were treated with a range of doses of the bioreductive drugs for a period of up to 48 h and the extent of cell kill was assessed at various times after treatment. Cytotoxicity was determined using a clonogenic cell-survival assay. The results demonstrated that two of the nitrofurans were even more toxic to spheroid cells than was the potent bioreductive nitroimidazole aziridine RSU 1069. The dose of the nitrofuran which, after a 24-h exposure, led to a survival value between 0.5 and 1.0, then was chosen for subsequent studies aimed at assessing the ability of these agents to potentiate the efficacy of the nitrosourea CCNU. Exposure to this chemotherapeutic agent was for a period of 1 h. The results indicated that all four dual function nitrofurans enhanced the cell killing of the conventional chemotherapeutic agent by factors ranging from 1.1 to 1.7. Subsequent studies evaluated the therapeutic benefit of combining these bioreductive agents and CCNU in KHT sarcoma-bearing C3H/HeJ mice. The nitrofurans were administered i.p. 0.5 h prior to the chemotherapy and tumor response was assessed by measuring the survival of clonogenic KHT cells 22–24 h after treatment. Normal tissue toxicity was determined using a bone marrow stem cell (CFU-GM) assay. Combining these bioreductive agents with CCNU increased the tumor cell kill by factors of 1.2 to 1.7. Bone marrow toxicity increased with CCNU dose, but the addition of the nitrofurans failed to enhance this endpoint of normal tissue toxicity.

Hypobaric hypoxia: A method for testing bioreductive drugs in vivo

Hypobaric hypoxia has been used to induce tumor hypoxia for in vivo comparison of the anti-tumor effects of the bioreductive agents SR 4233 (3-amino-1,2,4-benzotriazine-1,4-dioxide), RSU 1069 (1(2-nitro-1-imidazolyl)-3-aziridino-2-propanol), and Nitromin (methylbis(2-chloroethyl)amine N-oxide). BDF mice bearing the T50/80 mammary carcinoma were treated with these agents over a range of doses under normobaric (oxic) and hypobaric (hypoxic) conditions. The time taken for the tumor to double treatment volume (volume doubling time) was used as a measure of anti-tumor effect. Volume doubling time was plotted against log dose and dose response curves were fitted. A dose enhancement ratio (the ratio of drug doses required to give an equivalent anti-tumor effect under oxic and hypoxic conditions) was determined. The dose enhancement ratios for SR 4233 and RSU 1069 were 8.8 and 8.5, respectively, showing that these agents had an equivalent and substantial enhancement of their cytotoxicity when combined with hypobaric hypoxia. For Nitromin, no significant dose response effect was obtained under oxic conditions precluding the calculation of the dose enhancement ratio. SR 4233 was found to have increased systemic toxicity when combined with hypobaric hypoxia, suggesting that it is more readily activated than the other drugs tested. This in vivo test system will allow determination of the dose enhancement ratio for novel bioreductive agents and facilitate their comparison.

Evaluation of bioreductive drugs in multicell spheroids

The therapeutic potential of a variety of bioreductive agents, including misonidazole, RSU-1069, NFVO, mitomycin C, porfiromycin, and SR-4233 was evaluated using Chinese hamster V79 multicell spheroids in vitro. Fluorescenceactivated cell sorting techniques were used to selectively recover cells from various depths within the spheroids to measure the differential cytotoxicity in the cells near the hypoxic core of the spheroid relative to the well oxygenated peripheral cells. At the high cell density found in spheroids (as in tissues in vivo) the differential toxicity observed was typically much less than expected, based on data from single cell systems. In some cases, this was due to lack of sufficient hypoxia in the spheroids; in other cases, drug treatment itself produced reoxygenation through metabolic or toxic effects during treatment. An unexpected observation of considerable concern was rapid bioreduction of the more active agents; this sometimes occurred at rates that exceeded drug delivery, resulting in considerably less efficacy when large hypoxic fractions were present (e.g. mitomycin C, NFVO, and SR-4233). This suggests that induction of hypoxia prior to bioreductive agent therapy may not be the most productive approach. Though none of the agents showed “ideal” properties, porfiromycin was judged to give the best combination of differential toxicity, longevity in situ, and ability to reach the entire hypoxic cell subpopulation.

Pharmacokinetics, distribution, and metabolism of the novel bioreductive alkylating indoloquinone EO9 in rodents

The Indoloquinone E09 is a novel and potent bioreductive agent related in structure to mitomycin C but differing in many aspects of its antitumor activity, toxicity, and enzymatic activation. Because it is about to undergo clinical trial, we have investigated the pharmacokinetics of E09 in mice and rats. At the highest tolerated dose in male C3 H He mice (12 mg/kg iv) the initial plasma concentration (C 0) was 1.8 μg/ml. The drug was cleared rapidly with a t 1 2 of 1.9 min. The volume of distribution (Vd) was large (7.5 ml g −1) and the plasma clearance (Ci p) correspondingly high (2.6 ml g −1 min −1). The AUC o−x. was 4.8 μg ml −1 min. Equally rapid elimination was noted at the lower dose of 6 mg/kg iv. For comparison, the t 1 2 for the same dose of mitomycin C was much longer at 16 min and the peak plasma level 4-fold higher. In male Sprague-Dawley rats receiving 3 mg kg −1 the Co was 1.5 μg/ml and the t 1 2 was again short at 3.0 min. V d was 2.2 ml g ]−1, Cl p was 0.5 ml g −1min −1, and AUC o−x was 6.2 μg ml −1 min. No parent drug was detected in urine, but extensive biotransformation was confirmed by the detection of around 20% of the dose as metabolites, including the aziridine ring-opened hydrolysis product E05A. No drug or metabolite was detected in tumor or tissues. The results show that cytotoxic drug levels can be achieved for a short period in rodent plasma. The extremely fast excretion is consistent with the rapid rates of bioreductive metabolism in vitro. These data should be useful in the forthcoming clinical trials of E09, where a pharmacokinetically guided dose escalation may be used, and also in the design and development of second generation analogues.

Gene regulation of aromatic L-amino acid decarboxylase (AADC) by NSD-1015 in rat PC12 penochromocytoma cells

Tumor blood flow modification is currently under investigation as a possible means of optimizing current cancer therapies, with particular respect to improving the efficacy of bioreductive agents. A variety of calcium channel blockers have been shown to modify tumor perfusion in model systems, and may be valuable as potentiators of both bioreductive and conventional drugs. We report the effects of nifedipine, verapamil, flunarizine, and cinnarizine on renal function in C3H mice, assayed by clearance of simultaneously injected 51Cr ethylenedlamine tetraacetate. Nifedipine at 10 mgkg−1 blocked 51Cr ethylenediamine tetraacetate clearance for 30 min and reduced its subsequent rate of clearance by a factor (± 2 se) of 2.4 ± 0.6. At 1 mgkg−1 it reduced the rate of clearance by a factor of 1.2 ± 0.2. Verapamil at 10 mgkg−1 blocked 51Cr ethylenediamine tetraacetate clearance by a factor of 1.2 ± 0.2. Verapamil at 10 mgkg−1 blocked 51Cr ethylenediamine had no effect at 1 mgkg−1 Flunarizine had no effect at 50 mgkg−1 or at 5 mgkg−1 but cinnarizine at 50 mgkg−1 reduced clearance rate by a factor of 1.2 ± 0.1. The data show that some of these vasoactive agents, nifedipine and verapamil in particular, can severely compromise renal function and may, therefore, affect the plasma pharmacokinetics of co-administered drugs that are cleared by the kidney.