Hypoxic Cells In Solid Tumors Research Articles

Abstract PO-033: The angiotensin II type 1 receptor blocker telmisartan inhibits the development of transient tumour hypoxia and improves response to ionizing radiation therapy

Abstract Poorly oxygenated (hypoxic) cells are key components of the solid tumour microenvironment that drive tumour heterogeneity. Hypoxia regulates a variety of processes that promote tumour cell survival and metastasis, and hypoxic tumour cells are resistant to therapy. Patients with tumours that contain hypoxic cells have worse outcome after radiotherapy, chemotherapy, and even surgery, underscoring the therapeutic relevance of hypoxia in driving more aggressive tumour phenotypes. Hypoxic tumour cells result from deficiencies in the delivery of oxygen relative to the high oxygen demand of tumour cells. “Chronic” hypoxia develops due to the limited diffusion distance of oxygen through tumour tissue, while “transient” hypoxia occurs due to fluctuations in microregional tumour perfusion and oxygen delivery over time. Transiently hypoxic cells are more resistant to radiation therapy and are more metastatic than chronically hypoxic cells although, despite decades of work in this area, there are no effective long-term strategies to specifically target transiently hypoxic cells in solid tumours. Angiotensin II type 1 receptor (AT1R) blockers (ARBs) are widely prescribed anti-hypertensive agents that can also exhibit anti-fibrotic activity. Recent evidence indicates that the ARB losartan can inhibit collagen I (Col1) deposition in tumours by cancer-associated fibroblasts (CAFs), reduce blood vessel constriction, and increase net tumour blood flow, although the influence of losartan or other ARBs on transient hypoxia and tumour radiation response is unknown. We hypothesized that ARB-mediated inhibition of Col1 deposition from CAFs increases tumour blood flow by stabilizing microregional fluctuations in tumour perfusion and that ARBs will reduce transient tumour hypoxia. We tested the ARBs losartan and telmisartan in human tumour xenografts using positron emission tomography and intravenously injected fluorescent perfusion dyes to quantify tumour perfusion, and a combination of exogenous hypoxia markers and the hemorheological agent pentoxifylline to assess transient tumour hypoxia. We found that CAF-containing tumours had reduced Col1 levels in response to telmisartan, but not losartan, consistent with the improved pharmacokinetics and AT1R binding affinity of telmisartan. Telmisartan increased net tumour blood flow and stabilized microregional fluctuations in tumour perfusion. Importantly, telmisartan decreased overall hypoxia in the tumours by specifically targeting transient tumour hypoxia, which translated into a significant improvement in radiation response. These data indicate that telmisartan reduces a therapeutically-relevant population of transiently hypoxic tumour cells. Understanding how the solid tumour microenvironment influences cancer therapy is central to improving treatment outcome. Our work underscores the importance of transient tumour hypoxia and supports future clinical studies to evaluate telmisartan as a neoadjuvant for radiation therapy. Citation Format: Brennan J. Wadsworth, Rachel A. Cederberg, Che-Min Lee, Natalie S. Firmino, S. Elizabeth Franks, Jinhe Pan, Nadine Colpo, Kuo-Shyan Lin, Francois Benard, Kevin L. Bennewith. The angiotensin II type 1 receptor blocker telmisartan inhibits the development of transient tumour hypoxia and improves response to ionizing radiation therapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-033.

Preclinical study on hypoxic radiosensitizing effects of glycididazole in comparison with those of doranidazole in vitro and in vivo.

To overcome the radioresistance of hypoxic cells in solid tumor, numerous types of radiosensitizers specifically against them have been developed. Glycididazole has a chemical structure in which two metronidazole forms are combined, and is widely used as a hypoxic radiosensitizer in China. However, a detailed investigation of its radiosensitizing properties has not been performed. The present study reported a comparative assessment of glycididazole and doranidazole, another hypoxic radiosensitizer. All experiments were performed using the murine squamous cell carcinoma cell line SCCVII. Prior to X-irradiation, the cells were treated with the test drugs at concentrations of 10 mM and 200 mg/kg in vitro and in vivo, respectively. Uptake and their intratumor chemical forms were analyzed by high performance liquid chromatography (HPLC). Both drugs enhanced the reproductive cell death induced by X-irradiation under hypoxia. However, the growth delay assay of the transplanted tumor revealed the combination of X-irradiation and glycididazole showed a similar antitumor effect to that of X-irradiation alone, whereas doranidazole significantly sensitized the cells to X-irradiation. HPLC analysis revealed that incorporated glycididazole was decomposed to metronidazole and was therefore present at a lower concentration compared with that of doranidazole. The decomposition of glycididazole to metronidazole reduced its radiosensitizing efficiency in vivo. Elucidation of the kinetics of drugs containing metabolizable chemical forms is necessary for the optimization of clinical treatment.

The resveratrol analog HS-1793 enhances radiosensitivity of mouse-derived breast cancer cells under hypoxic conditions.

Tumor hypoxia is associated with treatment resistance, cell proliferation, and metastatic potential, all of which contribute to a poor prognosis. Resveratrol [RES (trans-3,4',5-trihydroxystilbene)], a naturally occurring polyphenol, is enriched in grapes and red wine. This study investigated whether the resveratrol analog HS-1793 modulates the hypoxic status and the level of perfusion in mouse breast cancer FM3A cells. Our data show that HS-1793 decreased the levels of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor protein under hypoxic conditions in FM3A cells. HS-1793 improved perfusion and hypoxic status in tumor tissues and inhibited angiogenesis through HIF-1α suppression in mice. Moreover, HS-1793 inhibited hypoxia-induced cancer stem cell properties and enhanced ionizing radiation-induced apoptosis in hypoxic FM3A cells. Collectively, the resveratrol analog HS-1793 might act as a potent radiosensitizer and be a useful adjuvant agent against radiotherapy-resistant hypoxic cells in solid tumors.

ET-32 * HYPOXIA REDUCTION IN INTRACRANIAL GLIOBLASTOMA MODELS BY OMX-4.80P, A PEGylated ENGINEERED H-NOX OXYGEN CARRIER THAT IS LONG-LASTING IN CIRCULATION AND SAFE

BACKGROUND: Hypoxic cells in solid tumors are resistant to radiotherapy. Omniox has developed a novel class of oxygen carrier proteins termed H-NOX, engineered to deliver oxygen selectively into hypoxic tissues. Previous studies have shown that one of our H-NOX candidates, OMX-4.80, penetrates intracranial glioblastoma (GB) tumors in mice, and in a dose dependent manner decreases tumor hypoxia and extends survival when combined with radiotherapy. However, we discovered that the circulation half-life of OMX-4.80 is ∼1h in dogs, which may limit clinical utility. To improve the circulation half-life, we developed a PEGylated version of OMX-4.80 (OMX-4.80P). The aim of this study was to characterize the pharmacokinetic and safety profile of OMX-4.80P and examine its effect on tumor hypoxia. METHODS: After intravenous administration of OMX-4.80P to mice and rats bearing GB tumors, we collected tumors, blood and plasma, and analyzed OMX-4.80P pharmacokinetics, immunogenicity and its effects on blood cell counts and chemistries. We evaluated OMX-4.80P bio-distribution by immunohistochemistry and its effect on tumor hypoxia by ELISA of endogenous (HIF-1a) and exogenous (pimonidazole) hypoxia markers. RESULTS: Compared to OMX-4.80, OMX-4.80P has an increased stability and circulation half-life (30h vs. 1h), significantly longer tumor accumulation (48h vs. 4h), and is not immunogenic. Furthermore, OMX-4.80P crosses the blood tumor barrier in several different intracranial rodent GB models and reduces HIF-1a and pimonidazole accumulation by ∼50%. Pharmacokinetic and toxicology studies in rodents and dogs showed that OMX-4.80P is well tolerated, and has a circulation half-life of ∼52h in dogs. The resulting exposure is expected to be sufficient for OMX-4.80P to accumulate in human GB tumors and reduce hypoxia. CONCLUSIONS: In summary, the increased retention of OMX-4.80P in circulation, the longer exposure achieved in solid tumors and efficiency in delivering oxygen into hypoxic tissues suggest OMX-4.80P is a promising IND candidate for radiation therapy enhancement in GB.

Bioreductive prodrugs as cancer therapeutics: targeting tumor hypoxia.

Hypoxia, a state of low oxygen, is a common feature of solid tumors and is associated with disease progression as well as resistance to radiotherapy and certain chemotherapeutic drugs. Hypoxic regions in tumors, therefore, represent attractive targets for cancer therapy. To date, five distinct classes of bioreactive prodrugs have been developed to target hypoxic cells in solid tumors. These hypoxia-activated prodrugs, including nitro compounds, N-oxides, quinones, and metal complexes, generally share a common mechanism of activation whereby they are reduced by intracellular oxidoreductases in an oxygen-sensitive manner to form cytotoxins. Several examples including PR-104, TH-302, and EO9 are currently undergoing phase II and phase III clinical evaluation. In this review, we discuss the nature of tumor hypoxia as a therapeutic target, focusing on the development of bioreductive prodrugs. We also describe the current knowledge of how each prodrug class is activated and detail the clinical progress of leading examples.

Novel aliphatic N-oxide of naphthalimides as fluorescent markers for hypoxic cells in solid tumor

A series of novel aliphatic N-oxide of naphthalimides ( A1–A5) were designed and prepared. The N–O group was firstly introduced into the amine side chain tailed to planar naphthalimide chromophore as hypoxic bioreductive marker. Fluorescence image analysis showed that the compounds could be used as potential markers for hypoxic cells (V79) in vitro especially for A1 with 17 times hypoxic-oxic fluorescence differential, which was probably due to the bis-bioreduction mechanism.

Inhibition of HIF-1α by the anticancer drug TAS106 enhances X-ray-induced apoptosis in vitro and in vivo

In a previous study, we showed that a novel anticancer drug, 1-(3-C-ethynyl-β-D-ribo-pentofuranosyl)cytosine (TAS106, ECyd) increased the antitumour efficacy of X-irradiation. However, its effects on hypoxic cells in tumours remain unclarified. Here, we show that TAS106 enhances the induction of apoptosis in X-irradiated human gastric adenocarcinoma MKN45 and MKN28 cells under hypoxia in vitro. At the same time, the accumulation of HIF-1α observed under hypoxia was shown to be decreased to the level of normoxia in the presence of 0.1 μM TAS106. To study the function of HIF-1α protein in apoptosis of hypoxic cells, we employed an HIF-1α reductive approach using its specific antisense oligodeoxynucleotide. The reduction of HIF-1α gene expression dramatically enhanced X-ray-induced apoptosis in hypoxic cells. In in vivo experiments in which MKN45 cells were transplanted into severe combined immunodeficient (SCID) mice, TAS106 (0.5 mg kg−1) suppressed HIF-1α expression and subsequently reduced the area of the hypoxic region in the tumour and enhanced the induction of apoptosis in the hypoxic region when combined with 2 Gy of X-irradiation. These results suggest the possibility that TAS106 acts as a potent radiosensitiser through the inhibition of HIF-1α expression and can be a useful agent against radiotherapy-resistant hypoxic cells in solid tumours.

Synthesis and Evaluation of Novel 8‐Oxo‐8H‐cyclopenta[a]acenaphthylene‐7‐carbonitriles as Longwavelength Fluorescent Markers for Hypoxic Cells in Solid Tumor.

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Synthesis and evaluation of novel 8-oxo-8H-cyclopenta[a]acenaphthylene-7-carbonitriles as long-wavelength fluorescent markers for hypoxic cells in solid tumor

Novel bioreductive and long-wavelength fluorescent markers for hypoxic cells in solid tumor, 9-isocyano-8H-acenaphtho[l,2-b]pyrrol-8-one with the side chain of 2-nitroimidazole, were designed, synthesized, and evaluated in V79 379A Chinese hamster cells in vitro. Compounds A2 and A4 showed good hypoxic–oxic fluorescence differential in vitro (V79 cells) by using fluorescence scan ascent.

Hypoxia-Regulated Expression of Attenuated Diphtheria Toxin A Fused with Hypoxia-Inducible Factor-1α Oxygen-Dependent Degradation Domain Preferentially Induces Apoptosis of Hypoxic Cells in Solid Tumor

Tumor cells in hypoxic areas of solid tumors are resistant to conventional chemotherapy and radiotherapy and thus are obstacles of cancer therapy. We report here the feasibility of applying hypoxia-regulated expression of diphtheria toxin A (DT-A) for killing hypoxic tumor cells. The expression vector was constructed to express DT-A fused with hypoxia-inducible factor-1alpha (HIF-1alpha) oxygen-dependent degradation (ODD) domain under the control of vascular endothelial growth factor gene promoter and contain erythropoietin mRNA-binding protein (ERBP)-binding sequence downstream of the DT-A/ODD sequence. In vitro ubiquitination assay showed that DT-A/ODD, but not DT-A, was ubiquitinated as efficient as HIF-1alpha under normoxic conditions in a von Hippel-Lindau- and oxygen-dependent manner. DT-A/ODD exhibited a comparable translation inhibitory activity to DT-A. ERBP-binding sequence was effective in stabilizing mRNA under hypoxic conditions in various cell types. Transfection of the vector expressing DT-A/ODD into high-metastatic Lewis lung carcinoma (3LL) A11 cells resulted in induction of apoptosis independently of hypoxia, probably due to its extreme toxicity. However, transfection of the vector expressing attenuated DT-A(W153F)/ODD or DT-A(H21A)/ODD resulted in a hypoxia-dependent induction of apoptosis. Liposomal gene transfer of the vector encoding DT-A(W153F)/ODD induced apoptosis in hypoxic, but not in normoxic, areas of solid tumors established by A11 variant cells with higher resistance to hypoxia-induced apoptosis and inhibited the growth of hypoxic tumors established by 3LL-P29 cells. These results suggest that hypoxia-regulated expression of attenuated DT-A(W153F)/ODD fusion protein is potentially of use for killing hypoxic tumor cells with minimizing the damage to normoxic normal tissues.

1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine: An anticancer agent targeting hypoxic cells

To target malignant cells residing in hypoxic regions of solid tumors, we have designed and synthesized prodrugs generating the cytotoxic alkylating species 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE) after bioreductive activation. We postulate that one of these agents, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119), requires enzymatic nitro reduction to produce 90CE, whereas another agent, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(4-nitrobenzyloxy)carbonyl]hydrazine (PNBC), can also be activated by nucleophilic attack by thiols such as glutathione (GSH)/GST. We demonstrated that these agents selectively kill hypoxic EMT6 mouse mammary carcinoma and CHO cells. In hypoxia, 50 microM KS119 produced 5 logs of kill of EMT6 cells without discernable cytotoxicity in air; similar effects were observed with CHO cells. PNBC was less efficacious against hypoxic tumor cells and also had some toxicity to aerobic cells, presumably because of GST/thiol activation, making PNBC less interesting as a selective hypoxic-cell cytotoxin. BALB/c mice with established EMT6 solid tumors were used to demonstrate that KS119 could reach and kill hypoxic cells in solid tumors. To gain information on bioreductive enzymes involved in the activation of KS119, cytotoxicity was measured in CHO cell lines overexpressing NADH:cytochrome b5 reductase (NBR), NADPH:cytochrome P450 reductase (NPR), or NADPH: quinone oxidoreductase 1 (NQO1). Increased cytotoxicity occurred in cells overexpressing NBR and NPR, whereas overexpressed NQO1 had no effect. These findings were supported by enzymatic studies using purified NPR and xanthine oxidase to activate KS119. KS119 has significant potential as a hypoxia-selective tumor-cell cytotoxin and is unlikely to cause major toxicity to well oxygenated normal tissues.

Utility of 19F MRS detection of the hypoxic cell marker EF5 to assess cellular hypoxia in solid tumors

Background and purposeThe present studies were undertaken to determine whether 19F MRS could be used to quantify the binding of the pentafluorinated derivative of etanidazole (EF5) in hypoxic cells of solid tumors. Materials and methodsA 4.7T imaging magnet was used for the in situ and in vitro evaluation of EF5 signals. In order to develop a better understanding of these NMR measurements the characteristics of parent, reduced unbound, and reduced bound EF5 signals were examined in vitro using a 12T spectrometer. ResultsIn situ data acquired using a 4.7T imaging magnet, showed retention of EF5 signals in KHT sarcomas that was absent in muscles for 6h after EF5 injection. In vitro studies showed no difference in the NMR detectable signal of parent and reduced unbound EF5. T2 values determined using parent EF5 samples revealed a T2 time of 675ms. In contrast, EF5 bound to KHT tumor cells gave rise to signals of low intensity, broad line widths, and T2 relaxation times of less than 30ms. When the same samples were analyzed using the 4.7T imaging magnet, the CF3 and CF2 fluorine peaks were readily identifiable in the parent EF5 sample but no fluorine signal could be detected from EF5 bound to KHT tumor cells. ConclusionThe inability to resolve bound EF5 metabolites even at high field strengths (12T), coupled with the short T2 relaxation times of the bound EF5, and the limits of detection of the in situ applied imaging magnet (4.7T), meant that hypoxic cells could not be quantified in tumors using the 19F MRS technique. In situ 19F MRS measurements of EF5 signals (parent/reduced unbound) may reflect conditions of tumor physiology and thus indicate the extent of tumor hypoxia but they are not capable of resolving the cellular oxygenation status of the tumor cells.

Prospects for hypoxia-activated anticancer drugs.

The occurrence of hypoxic cells in solid tumors, and their resistance to radiotherapy and many chemotherapeutic drugs, has engendered an interest in non-toxic prodrugs that can be activated selectively under hypoxic conditions. Despite this, no such compounds are yet registered for clinical use, due to the difficulty of their design and of measuring the extent of hypoxia clinically, and the failure of early examples. A new appreciation of the critical importance of the extravascular diffusion of the parent prodrug from the blood vessels to the remote hypoxic cells, and the back-diffusion of the activated cytotoxin from the hypoxic cells to surrounding tumor cells, is now guiding drug design in this area. New principles for the selective activation of prodrugs have also been reported, including using the reducing species generated in cells by radiotherapy itself, and using non-pathogenic anaerobic bacteria as a hypoxia-dependent vector for the delivery of prodrug-activating enzymes in a suicide gene therapy context.

Oxidation of 2-deoxyribose by benzotriazinyl radicals of antitumor 3-amino-1,2,4-benzotriazine 1,4-dioxides.

Tirapazamine (3-amino-1,2,4-benzotriazine 1,4-dioxide) is the lead bioreductive drug in clinical trials as an anticancer agent to kill refractory hypoxic cells of solid tumors. It has long been known that, upon metabolic one-electron reduction, tirapazamine induces lethal DNA double strand breaks in hypoxic cells. These strand breaks arise from radical damage to the ribose moiety of DNA, and in this pulse radiolysis and product analysis study we examine mechanistic aspects of the dual function of tirapazamine and analogues in producing radicals of sufficient power to oxidize 2-deoxyribose to form radicals, as well as the ability of the compounds to oxidize the resulting deoxyribose radicals to generate the strand breaks. Both the rate of oxidation of 2-deoxyribose and the radical yield increase with the one-electron reduction potentials of the putative benzotriazinyl radicals formed from the benzotriazine 1,4-dioxides. Subsequent oxidation of the 2-deoxyribose radicals by the benzotriazine 1,4-dioxides and 1-oxides proceeds through adduct formation followed by breakdown to form the radical anions of both species. The yield of the radical anions increases with increasing one-electron reduction potentials of the compounds. We have previously presented evidence that oxidizing benzotriazinyl radicals are formed following one-electron reduction of the benzotriazine 1,4-dioxides. The reactions reported in this work represent the kinetic basis of a short chain reaction leading to increased oxidation of 2-deoxyribose, a process which is dependent on the one-electron reduction potential of the benzotriazinyl radicals that are above a threshold value of ca. 1.24 V.

Inhibitory effect of a naphthazarin derivative, S64, on heat shock factor (Hsf) activation and glutathione status following hypoxia.

The presence of hypoxic cells in solid tumors has long been considered a problem in cancer treatment. Resistance of hypoxic cells to ionizing radiation and anticancer drugs has in part been attributed to changes in altered gene expression by hypoxia. We previously reported an activation of heat shock factor (Hsf) in murine tumor RIF cells following hypoxia and suggested that a subsequent accumulation of heat shock protein(s) (Hsp) is likely to contribute to the malignant progression of hypoxic tumor cells (Baek et al., 2001). In this study, we showed that hypoxia induced a DNA-binding activity of Hsf and activation of hsp70 gene expression in colon cancer Clone A cells, and that a naphthazarin derivative, S64, significantly inhibited the hypoxia-inducible hsp70 gene expression in Clone A cells. We also showed that S64 significantly reduced the cellular glutathione levels in this cell line. Considering the proposed effects of Hsp and glutathione on radiation and chemotherapy sensitivity, we suggest that the inhibitory effects of S64 on Hsf activation and cellular glutathione levels have potentially important clinical implications. We believe that the previously reported in vitro and in vivo anti-tumor effect of S64 (Song et al., 2000a, 2001) might be attributed, at least in part, to its effect on Hsf activation and/or glutathione depletion. We also believe that the detailed molecular mechanisms underlying the effects of S64 on Hsf and glutathione level following hypoxia deserve a more rigorous future study, the results of which could offer novel strategy to manipulate the resistance mechanisms of solid tumors.

69 disc Turnover rate of hypoxic cells in solid tumors

69 disc Turnover rate of hypoxic cells in solid tumors

Synthesis and evaluation of two technetium-99m-labeled peptidic 2-nitroimidazoles for imaging hypoxia.

The presence of hypoxic cells in solid tumors is a marker for therapy-resistant, aggressive disease. The noninvasive detection of hypoxic cells in tumors by radiolabeled 2-nitroimidazoles is a diagnostic technique under current evaluation. Two peptidic agents, dimethylglycyl-L-seryl-L-cysteinyl-lysyl{N(epsilon)-[1-(2-nitro-1H -im idazolyl)acetamido]}glycine (RP435) and dimethylglycyl-tert-butylglycyl-L-cysteinyl-glycine-[2-(2-ni tro-1H-im idazolyl)ethyl]amide (RP535) have been synthesized. Both agents contain an N(3)S class chelator for (99m)Tc and Re and a 2-nitroimidazole group which can be enzymatically reduced and selectively trapped in cells under hypoxic conditions. Two isomers of (99m)TcO-RP435, which are assumed to be syn and anti conformations, were observed on HPLC analysis. The interconversion of the two isomers in aqueous solution was investigated. In contrast, RP535 chelated (99m)Tc to form a single isomer and no conversion to its counterpart has been observed on HPLC analysis. The tert-butyl group on the chelator may inhibit the formation and interconversion of the syn and anti isomers of (99m)TcO-RP535. Both tracers showed a significant degree of hypoxia-specific accumulation in an in vitro assay, with (99m)TcO-RP535 showing higher selectivity for hypoxic cells than (99m)TcO-RP435. These results suggest that (99m)TcO-RP535 represents a lead compound worthy of further investigation as an agent for imaging hypoxia in tumors.

Hypoxia-selective nitrobenzyloxycarbonyl derivatives of 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazines.

Some 4- and 2-(nitrobenzyloxycarbonyl)-1, 2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazines (4, 6, and 7) were synthesized and evaluated for their ability to exert preferential toxicity to hypoxic EMT6 mammary carcinoma cells using a colony-forming assay. Of these, the 4,5-dimethoxy-2-nitro analogue 6 (50 microM, 1-h exposure) caused greater than 3 logs of kill of hypoxic cells, with relatively minor toxicity to corresponding aerobic cells. The ability of 4-nitro (4) and 4,5-dimethoxy-2-nitro (6) analogues to reach and kill hypoxic cells of solid tumors was also demonstrated using intradermally implanted EMT6 solid tumors in mice. In addition, a possible source of toxicity to normal tissue, i. e., the activation of the 4-nitrobenzyl derivative 4 by glutathione S-transferase-catalyzed thiolysis, was essentially eliminated by replacing one of the benzylic methylene protons by a methyl group. The 4-nitro (4) and 4,5-dimethoxy-2-nitro (6) analogues also appear to be reduced more easily under acidic conditions (pH 6.0) than under neutral conditions, as measured by differential pulse polarography. Since the pH in hypoxic regions is often lower than that in adjacent aerobic regions, this property should aid in the cytotoxic action of these agents against hypoxic cells of solid tumors.

Studies of 99mTc-BnAO (HL-91): a non-nitroaromatic compound for hypoxic cell detection

Purpose: Solid tumours of similar type and stage can vary widely in their hypoxic cell fraction. Such cells may be prognostic for aggressive, metastatic, and radiation-resistant disease. A 99mtechnetium ( 99mTc)-labelled non-nitroaromatic agent, butyleneamine oxime ( 99mTc-BnAO) or HL-91 (Amersham International, Inc., Amersham, UK) has been evaluated both in vitro and in vivo for its possible efficacy as a noninvasive marker for the clinical detection of hypoxic cells in solid tumours. Materials and Methods: Suspension cultures of Chinese hamster ovary (CHO) cells under controlled levels of oxygen were used to measure the oxygen dependency of 99mTc-BnAO accumulation. V79 cells grown as multilayers on a semipermeable membrane served as an in vitro model for drug penetration through the extravascular space of the tumour. C3H mice bearing KHT-C leg tumours were the in vivo models for selective drug accumulation as a function of time after i.v. administration of 99mTc-BnAO. Results: 99mTc accumulated selectively in hypoxic vs. aerobic cells, resulting in a 9 ± 2-fold differential in radioactivity per cell at 4 h. The k m for this selective accumulation was 20 ppm of oxygen. The labelled drug was equally effective in penetrating the cellular multilayer under aerobic or hypoxic conditions. In vivo measurements indicated favourable labelling of solid tumours containing hypoxic cells with 1% of the total activity per g of tumour, a tumour-to–blood ratio of 1.2, and a tumour-to–muscle ratio of 4.6 at 4 to 6 h after drug administration. In contrast to more lipophilic 99mTc- labelled compounds, excretion was primarily via the urinary tract. Nitro- L-arginine selectively increased solid tumour labelling over normal tissue. Conclusions: 99mTc-BnAO or HL-91 is a promising agent for clinical studies of tumour hypoxia, although the mechanism of its selective hypoxic cell accumulation remains unexplained.

Reduction of hypoxic cells in solid tumours induced by mild hyperthermia: special reference to differences in changes in the hypoxic fraction between total and quiescent cell populations.

C3H/He mice bearing SCC VII tumours received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps in order to label all proliferating (P) cells. The tumours were then heated at 40 degrees C for 60 min. At various time points after heating, tumour-bearing mice were irradiated while alive or after being killed. Immediately after irradiation, the tumours were excised, minced and trypsinized. The tumour cell suspensions obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labelling, which could be regarded as quiescent (Q) cells, was determined using immunofluorescence staining for BrdU. The MN frequency in the total (P+Q) tumour cell population was determined from the irradiated tumours that were not pretreated with BrdU. The MN frequency of BrdU unlabelled cells was then used to calculate the surviving fraction of the unlabelled cells from the regression line for the relationship between the MN frequency and the surviving fraction of total (P+Q) tumour cells. In general, Q cells contained a greater hypoxic fraction (HF) than the total tumour cell population. Mild heating decreased the HF of Q cells more markedly than in the total cell population, and the minimum values of HFs of both total and Q cell populations were obtained 6 h after heating. Two days after heating, the HF of total tumour cells returned to almost that of unheated tumours. In contrast, the HF of Q cells did not return to the HF level of unheated tumours until 1 week after heating. It was thought that irradiation within 12 h after mild heating might be a potentially promising therapeutic modality for controlling radioresistant Q tumour cells.