Fused pyrazine mono-n-oxides as bioreductive drugs. II Cytotoxicity in human cells and oncogenicity in a rodent transformation assay.

PURPOSE: To determine what structural moieties of the fused pyrazine mono-N-oxides are determining factors in their in vitro cytotoxicity and oncogenicity. METHODS AND MATERIALS: A new series of experimental bioreductive drugs, fused pyrazine mono-N-oxides, was evaluated in vitro for aerobic and hypoxic cytotoxicity in the HT29 human colon adenocarcinoma cell line by using clonogenic assays. The relative oncogenicities of these compounds were also determined in aerobic cultures of C3H 10T1/2 mouse embryo fibroblasts by using a standard transformation assay. RESULTS: Removal of the 4-methyl piperazine side chain from the parent compound, RB 90740, reduced the potency of the hypoxic cytotoxin. Reduction of the N-oxide function increased the aerobic cytotoxicity and eliminated most of the hypoxic/aerobic cytotoxic differential. The reduced N-oxide also had significant oncogenicity, consistent with a mechanism of genotoxicity following bioreduction of RB 90740. CONCLUSION: This new series of bioreductive compounds may be effective in cancer therapy, particularly the lead compound RB 90740. The oncogenic potential of these compounds is similar to that for other cancer therapies. Further studies should include evaluation of these compounds in vivo and the development of analogs with reduced oncogenic potential and retention of the hypoxic/aerobic cytotoxicity differential.

Synergistic interaction between tirapazamine and cyclophosphamide in human breast cancer xenografts.

This study examined the efficacy of combining cyclophosphamide and the hypoxic cytotoxin, tirapazamine, in the treatment of human breast cancer xenografts grown in nude mice. A single dose of tirapazamine was followed 2 h later by a single dose of cyclophosphamide. As determined from tumor regrowth delay, the effectiveness of combined therapy was greater than the additive effects of each treatment given alone. Possible mechanisms of this synergistic interaction include enhancement of DNA damage, inhibition of repair of DNA damage, or induction of apoptosis. Apart from some loss in body weight, the only other toxicity of interest in mice treated with tirapazamine was necrosis of the skin on the distal tail, which appeared to be vascular in origin.

Local hyperthermia and SR 4233 enhance the antitumor effects of radioimmunotherapy in nude mice with human colonic adenocarcinoma xenografts.

Local hyperthermia and the hypoxic cytotoxin SR 4233 were administered to nude mice with 693 +/- 47 mm3 (mean +/- SE) s.c. HCT-8 human colonic adenocarcinoma xenografts in an attempt to enhance the antitumor effects of radioimmunotherapy. Biodistribution studies revealed preferential binding of NR-Lu-10, a murine monoclonal antibody, to the tumors compared with an isotype-matched control antibody, CCOO16-3.A single injection of 25 microCi 90Y-NR-Lu-10 significantly inhibited tumor growth (control versus 90Y-NR-Lu-10: P = 0.048). The administration of hyperthermia at 41.5 degrees C for 1 h immediately following the injection of 111In-labeled NR-Lu-10 up-regulated tumor-associated antigen expression and increased antibody uptake in the tumors by 73% (P = 0.001) without significantly affecting antibody uptake in normal tissues. However, the heat treatment did not produce a more homogeneous distribution of the antibodies in the tumors and did not significantly enhance the tumor growth delay produced by 90Y-NR-Lu-10 (P = 0.07). The administration of local hyperthermia at 43.0 degrees C for 1 h, on the other hand, had direct cytotoxic effects (P = 0.03) and enhanced the tumor growth delay produced by 90Y-NR-Lu-10 (P = 0.01). SR 4233 also enhanced the tumor growth delay produced by 90Y-NR-Lu-10 (P = 0.03). The greatest antitumor effects were observed when both hyperthermia at 43.0 degrees C and SR 4233 were administered in combination with 90Y-NR-Lu-10 (P = 0.002). No toxicity was produced by the local hyperthermia, and the only toxicities produced by 90Y-NR-Lu-10 and SR 4233 were neutropenia and weight loss.

Experimental radioimmunotherapy.

Radiolabeled monoclonal antibodies have been used for radioimmunotherapy studies with human tumor spheroids and murine and human tumor xenografts in experimental animals. This paper reviews the work that has been performed in these models with different types of cancer, and highlights those papers that have presented dosimetry estimates and attempts to correlate the findings. Radioimmunotherapy studies in multicell spheroids, as a model for micrometastases, have been performed in human neuroblastoma, colon cancer, and melanoma cell lines using 131I-, 125I-, 186Re-, and 212Bi-labeled antibodies. The uniform geometry of the spheroid has allowed radiation dose estimates to be made. Up to three logs of cell kill have been achieved with 131I- and 186Re-specific antibody with minimal toxicity from labeled nonspecific antibody, but 212Bi-antibody had little effect because of its short half-life as shown by Langmuir. It appears that the two most important factors for therapeutic efficacy in this model are good penetration of the radiolabeled antibody and an adequate radionuclide half-life to allow penetration of the immunoconjugate prior to significant radionuclide decay. Radioimmunotherapy studies in animals bearing transplants of colon cancer, leukemia, lymphoma, hepatoma, renal cell carcinoma, neuroblastoma, glioma, mammary carcinoma, small cell lung carcinoma, cervical carcinoma, ovarian carcinoma, and bladder cancer have been performed with 131I, 90Y, 186Re, 153Sm, and 177Lu beta emitting, and 212Bi alpha emitting radionuclides conjugated to monoclonal antibodies. A few studies compared different radionuclides in the same model system. The approaches that have been used in these studies to estimate tumor dosimetry include the MIRD approach, thermoluminescent dosimetry, autoradiography, and comparison to external irradiation. The majority of investigators have estimated the dose to tumor and normal organs using MIRD-based calculations (time-activity curve and equilibrium dose constant method). The range of tumor doses has been between 17 and 11 171 mGy/MBq of administered radioactivity. The effectiveness of radiolabeled monoclonal antibody therapy depends on a number of factors relating to the antibody such as specificity, affinity, and immunoreactivity. The density, location, and heterogeneity of expression of tumor-associated antigen within tumors will affect the localization and therapeutic efficacy of radiolabeled antibodies, as will physiological factors such as the tumor vascularity, blood flow, and permeability. These factors are discussed and examples are presented.(ABSTRACT TRUNCATED AT 400 WORDS)

Radiobiology of radiolabeled antibody therapy as applied to tumor dosimetry.

This paper reviews the radiobiological aspects of radioimmunotherapy (RIT) with radiolabeled antibodies, including comparisons between RIT and external beam irradiation. The effectiveness of cell killing by radiation decreases with the dose rate and the rate of decrease is determined by the size of the shoulder on the radiation survival curve. Tumors with poor repair capabilities exhibit less of a dose rate effect than tumors with good repair capabilities. Continued tumor cell proliferation during treatment occurs at very low dose rates and can contribute to the reduced effectiveness of low dose rate radiation. Toxicity to normal tissues will determine the total dose of radiolabeled antibody that can be given and this will be influenced by the choice of both the radionuclide and the antibody. The reported enhanced effectiveness of RIT may be due to multiple factors including selective targeting of cells responsible for tumor volume doubling, tumor surface binding rather than homogeneous binding throughout the tumor volume, targeting of the tumor vasculature, or block of cell cycle progression in G2. During RIT, there is less time for reoxygenation of hypoxic tumor cells than during a course of conventional external beam radiotherapy. It has not yet been determined whether this will have a detrimental effect on RIT. Probably the most important factor in the success of RIT is dose heterogeneity. Any viable portion of a tumor that is not targeted and does not receive a significant radiation dose will potentially lead to treatment failure, no matter how high the dose received by the remainder of the tumor. Comparisons between RIT and external beam radiation have shown a wide range of relative efficacy. Tumors most likely to respond to RIT are tumors with poor repair capabilities, tumors that are susceptible to blockage in radiosensitive phases of the cell cycle, tumors that reoxygenate rapidly, and tumors that express the relevant antigen homogeneously. From a radiobiological perspective, it appears that RIT alone is unlikely to cure many tumors and that combination with other treatment modalities will be essential.

The combined use of 131I-labeled antibody and the hypoxic cytotoxin SR 4233 in vitro and in vivo.

Radioimmunotherapy is hindered by the slow penetration of antibody molecules into tumors. Cells that are poorly targeted by antibody, because of their distance from feeding blood vessels, receive the lowest radiation dose, and this problem is compounded if there are radioresistant hypoxic cells present. It would be desirable to combine radioimmunotherapy with an agent that is preferentially toxic to these cells. SR 4233 is a potent hypoxic cytotoxin, and it was combined with 131I-NR-LU-10 to treat LS174T human colon adenocarcinoma multicell spheroids and nude mouse xenografts for these studies. Under conditions of severe hypoxia (< 0.01% O2), 2 h of pretreatment or 18 h of simultaneous treatment with SR 4233 did not significantly enhance the effectiveness of 131I-NR-LU-10 in spheroids. However, under aerobic conditions with a 10% fraction of hypoxic cells, there was more toxicity than would be predicted from simple additivity. Xenografts treated with 131I-NR-LU-10 + SR 4233 had a growth delay that was significantly longer than that achieved with 131I-NR-LU-10 alone. In both spheroids and xenografts, combined treatment produced about 10 times more cell killing than 131I-NR-LU-10 alone. The lack of enhancement in spheroids under complete hypoxia suggests that SR 4233 does not sensitize hypoxic cells to radiation damage. The results with aerobic spheroids and in vivo, where a portion of the cells were hypoxic, could be explained by the targeting of different cell populations (hypoxic and aerobic) by each therapeutic modality. This effect should also be enhanced by reoxygenation and reestablishment of the hypoxic fraction during treatment, thus allowing more than the initially hypoxic fraction of cells to be killed by the SR 4233.

Comparisons between two monoclonal antibodies that bind to the same antigen but have differing affinities: uptake kinetics and 125I-antibody therapy efficacy in multicell spheroids.

It has been predicted that low affinity antibodies (Abs) should penetrate into tumors more readily than high affinity Abs. However, the absolute uptake and residence time of a high affinity Ab may be better. It is, therefore, not clear whether a high affinity Ab would have a therapeutic advantage. This is particularly relevant with 125I radioimmunotherapy, where targeting of every cell is important. This study compared the uptake kinetics and toxicity in multicell spheroids of two murine monoclonal Abs labeled with 125I. 17-1A was produced by immunization with a human colon cancer cell line and has an affinity of 5.15 x 10(7) M-1. 323/A3 was produced by immunization with a human breast cancer cell line and has an affinity of 1.87 x 10(9) M-1. Binding of both Abs to LS174T spheroids was similar at 4 degrees C, but binding of 17-1A was 8-10-fold less than that of 323/A3 at 37 degrees C. Despite this difference, the toxicity of 125I-17-1A in spheroids after 7 days of incubation was similar to that of 125I-323/A3. Autoradiography showed that 17-1A penetrated the spheroids much more deeply and evenly than did 323/A3. It appears that much of the radiation dose to spheroids treated with 125I-323/A3 was wasted because of the uneven Ab distribution. This study demonstrates the potential advantage of using Abs of lower affinity for 125I radioimmunotherapy, because of their more even distribution. It also suggests that a large number of binding sites per cell may be a disadvantage if more 125I is bound than is necessary to kill the cell, because this may slow Ab penetration.

Comparisons of sectioned micro-TLD dose measurements with predicted dose from 131I-labeled antibody.

The dose distribution from radioimmunotherapy is very heterogeneous because of variability in antigen expression, antibody penetration, and tumor architecture. Many models of dose distribution have been constructed but it has been very difficult to confirm these predictions with actual measured doses. The purpose of this study was to determine what degree of resolution could be obtained using mini-thermoluminescent dosimeter(s) (TLD) in a micrometastasis model. TLDs were inserted into 1-mm-diam multicell tumor spheroids that had been treated with 131I-labeled antibody. The spheroids were then sectioned at 30-microns intervals and the TLD sections (measuring 0.14 x 0.1 x 0.03 mm) were removed and read. Calibration of the TLDs was done with whole TLDs using external beam radiation and an 131I-containing gel, and with TLD sections using external beam radiation. Predicted doses were determined by measuring the activity in individual spheroids and the time the TLDs were in the spheroids and incorporating these numbers into a model that assumed either surface binding of 131I or some degree of penetration. There was a correlation between the measured TLD dose and the predicted absorbed dose when comparing the low- to high-dose regions. However, there was considerable variation within any particular dose range, probably due to heterogeneity in TLD grain size.(ABSTRACT TRUNCATED AT 250 WORDS)

Radioimmunotherapy: clinical results and dosimetric considerations.

Radiolabeled antibodies for cancer therapy are being investigated in clinical trials in more than 30 centers. 131Iodine-labeled antibody (Ab) therapy of solid tumors has produced few responses when given alone. When given in conjunction with chemotherapy and external beam therapy in hepatoma patients, objective responses have occurred. Because of the short range of 131I, 90Y and 186Re are being studied and objective responses have occurred in patients without the addition of other therapies. 131I-labeled Ab therapy of lymphoma, a radioresponsive tumor, has produced a much higher objective response rate than in other solid tumors. Regional RIT has not been shown to offer a definite advantage over the intravenous route. Tumor doses have generally been less than 2000 cGy per treatment with some tumors receiving higher doses. The bone marrow is the dose-limiting organ for RIT and marrow cryopreservation with subsequent reinfusion may prove useful.

Comparisons of the efficacy of 186Re- and 131I-labeled antibody in multicell spheroids.

Several radionuclides are being studied for use in radioimmunotherapy. Although 131I has been used most widely, there are several disadvantages to it including its large gamma-ray component, its rather long half-life, and its modest beta-particle energy. However, the beta-particle energy can be an advantage in very small tumors (less than 1-2 mm). 186Re has several potential advantages over 131I but it has never been directly compared with it experimentally. Dosimetry modeling predicted that 186Re would have a dose advantage over 131I at large tumor sizes but for tumors as small as 1 mm diameter, this advantage would be lost. In order to confirm these predictions experimentally, this study compared the relative efficacy of a pancarcinoma antibody, NR-LU-10 labeled with 186Re or 131I in 0.8-1.0 mm diameter LS174T human colon adenocarcinoma multicell spheroids. Spheroids were incubated for 90 hr at 37 degrees C and evaluated with clonogenic assay, autoradiography, and histology. When corrected for cumulative activity bound, both radionuclides were equally effective. Autoradiography demonstrated poor penetration of radionuclide into the depths of the spheroids. Because 186Re has a theoretical dose advantage in larger tumors and because it has been shown to be equivalent to 131I in tumors as small as 1 mm diameter, it may be superior to 131I in most clinical situations. However, in the treatment of micrometastases of less than 1 mm diameter, theoretical dosimetry modeling predicts that 131I or radionuclides with similar beta particle energies should be more effective.

The effect of antigen concentration, antibody valency and size, and tumor architecture on antibody binding in multicell spheroids.

Intact IgG1 and F(ab')2 anti-carcinoembryonic antigen antibodies penetrate human colon adenocarcinoma multicell spheroids much more slowly than Fab fragments and the molecular weight and the binding site valency appear to be the most important factor in determining the rate of penetration. The rate is also influenced considerably by the number of antigen binding sites per cell, with a high antigen concentration slowing penetration appreciably. The tumor cell architecture appears to have a minor effect on antibody penetration when compared to antibody size or antigen concentration.

Iodine-125-NRLU-10 kinetic studies and bismuth-212-NRLU-10 toxicity in LS174T multicell spheroids.

Alpha emitter-labeled antibodies (Abs) are of considerable interest in cancer therapy. Alpha particles are densely ionizing and therefore have a high radiobiologic effectiveness, and the cell killing produced is influenced very little by dose rate or hypoxic conditions. LS174T human colon adenocarcinoma spheroids were used in this study to evaluate the efficacy of alpha emitter-labeled Abs in a three-dimensional model. NRLU-10, an IgG2b Ab to a pancarcinoma antigen, and its Fab fragment were used. Initial kinetic studies using 125I-NRLU-10 revealed that a large number of binding sites/cell and high Ab affinity led to slow Ab penetration. This effect could be overcome by increasing the Ab concentration ten-fold for Fab but not for intact Ab. Bismuth-212-NRLU-10 therapy was very effective in killing single cells (over 3 log reduction in surviving fraction) but was ineffective in spheroids (less than 1 log reduction). This was likely due to inadequate penetration into the spheroids before the 212Bi decayed. The use of higher Ab concentrations, tumors with fewer antigenic sites/cell for the Ab being used, lower affinity Abs, alpha emitters with longer half-lives, and pretargeting with bifunctional Ab are all potential ways of increasing the efficacy of alpha emitter-labeled Abs for cancer therapy.

131I-anticarcinoembryonic antigen therapy of LS174T human colon adenocarcinoma spheroids.

LS174T human colon adenocarcinoma multicell spheroids were used to study the radiobiological aspects of radioimmunotherapy. The spheroids were incubated in 131I-anticarcinoembryonic antigen (B7) at an antibody concentration of 0.5 microgram/ml and at 131I concentrations of 2.5 and 7.5 microCi/ml. After incubation times of 90 h, clonogenic cells per spheroid were reduced by 1400-fold and 23-fold at the high and low 131I concentrations, respectively. 131I Nonspecific antibody (PX63) resulted in 2- and 1.2-fold reductions. Spheroid diameter was not significantly affected by therapy but histological examination revealed that there had been a significant reduction in the cell density, particularly near the spheroid surface. Using a theoretical model to estimate radiation dose, a radiation survival curve was constructed. The resulting curve was somewhat concave suggesting the presence of a resistant population of cells. It is likely that this observation is primarily due to the fact that the inner cells received a lower dose than the outer cells. A population of radiobiologically hypoxic cells in the inner portion of the spheroids may also have contributed to the decreasing slope of the curve as well as ongoing cell division leading to new cells which receive a lower radiation dose per cell cycle. Because of the ability to estimate radiation dose for a given biological effect, these types of experiments may allow predictions of the efficacy of radiolabeled antibody therapy for micrometastatic disease.

Fine needle aspiration cytology in the management of palpable benign and malignant breast disease. Correlation with clinical and mammographic findings.

Two surgeons in a health maintenance organization group practice performed 280 fine needle aspirations (FNAs) on 257 palpable breast lesions in 200 patients. The cytology was interpreted by four pathologists at a community hospital. FNA cytology had a sensitivity of 96% and a specificity of 94% in these cases; there were no false-positive diagnoses of cancer. FNA cytology improved the identification of benign disease and decreased the risk of missing cancers. FNA cytology also improved the predictive value of mammographic information and was more helpful than mammography in demonstrating the need for biopsy of small palpable lesions that turned out to be "minimal" breast cancers.

Dosimetry models for radioimmunotherapy.

Tumor therapy using radiolabeled antibodies presents a challenging problem in absorbed dose determination. The purpose of this study is to evaluate the effect of tumor size on the absorbed dose distribution from beta-emitters when the radiolabeled antibody is not uniformly distributed throughout the tumor. Two theoretical dosimetry models are constructed, one for nonvascularized micrometastases and the other for vascularized tumors. All calculations assume no penetration of radionuclide into the tumor. These are compared to an even distribution of radionuclide throughout the tumor. In micrometastases of 1-mm diameter or less, emitters of low energy such as 131I give higher dose rates than emitters of higher energy because less energy is lost outside the target volume. However, even with 131I, a significant proportion of the energy is not absorbed in the tumor and, as a result, the concentration of radionuclide necessary for a therapeutic radiation dose becomes higher as the tumor diameter gets smaller. Because it may be impossible to achieve these concentrations in very small tumors (less than 0.5-mm diameter), alpha-emitters may be useful in combination with beta-emitters for therapy of micrometastatic disease. In vascularized tumors, higher energy emitters such as 90Y yield higher doses because of overlapping dose distributions from multiple vascular sources. This also produces a more even dose distribution across a tumor, even when there is poor penetration of the radiolabeled antibody. Thus tumor size, antibody penetration, and tumor vascularity all influence the choice of radionuclide and, depending on the circumstances, alpha-emitters, low-energy beta-emitters, high-energy beta-emitters, or some combination of the three may be most efficacious.

Use of the pulmonary artery for left ventricular venting during cardiac operations.

Data relating to the hemodynamic efficaciousness and mechanism of action of a pulmonary artery catheter or vent used for left ventricular venting during cardiac operations are presented. The pulmonary artery vent is a plastic sump catheter that is introduced into the main pulmonary artery through a purse-string suture and connected via a roller pump to the venous reservoir of the heart-lung perfusion machine. Placement and removal require only a few minutes. The pulmonary artery vent retrieved 85% of a 99mtechnetium-labeled solution placed in the left atrium during aortic cross-clamping, and there was no detectable radioactivity in peripheral or aortic root blood samples. Pulmonary artery vent return during cardiopulmonary bypass in 10 patients undergoing coronary artery bypass averaged 12.5 L. The effectiveness of left ventricular decompression was evaluated in 20 patients also undergoing bypass grafting. Use of the pulmonary artery vent consistently and significantly decreased left heart pressures, compared to the control situation with the vent off, with the aortic cross-clamp applied, and in both the fibrillating and beating heart in the early postischemic reperfusion period. We reached the following conclusions: (1) The pulmonary artery vent withdraws left heart blood via the pulmonary vasculature, in addition to returning right heart spillover and retrieving bronchial flow. (2) Left heart pressures are reduced to levels which reduce oxygen demands and preserve endocardial perfusion, therefore protecting myocardium, during fibrillation and during coronary reperfusion of the beating heart. (3) Because of its effectiveness and safety, especially the impossibility of introducing air into the left ventricle, the pulmonary artery vent is recommended for routine left ventricular venting.