Dietmar W. Siemann

Pathophysiologic Effects of Vascular-Targeting Agents and the Implications for Combination with Conventional Therapies

A functional vascular supply is critical for the continued growth and development of solid tumors. It also plays a major role in metastatic spread of tumor cells. This importance has led to the concept of targeting the vasculature of the tumor as a form of cancer therapy. Two major types of vascular-targeting agent (VTA) have now emerged: those that prevent the angiogenic development of the neovasculature of the tumor and those that specifically damage the already established tumor vascular supply. When used alone neither approach readily leads to tumor control, and so, for VTAs to be most successful in the clinic they will need to be combined with more conventional therapies. However, by affecting the tumor vascular supply, these VTAs should induce pathophysiologic changes in variables, such as blood flow, pH, and oxygenation. Such changes could have negative or positive influences on the tumor response to more conventional therapies. This review aims to discuss the pathophysiologic changes induced by VTAs and the implications of these effects on the potential use of VTAs in combined modality therapy.

Modification of chemotherapy by nitroimidazoles

The potentiation of chemotherapeutic agents by radiation sensitizers has been extensively studied for several years. There is little doubt that the effectiveness of certain anti-cancer drugs, primarily alkylating agents, can readily be enhanced both in vitro and in vivo through the addition of a sensitizer. While enhanced effects have been observed in certain critical normal tissues, in general most animal model studies have demonstrated a therapeutic gain at large sensitizer doses. This approach to combination therapies therefore appears promising. Yet many questions concerning the interaction between chemotherapeutic agents and radiosensitizers, particularly in the area of mechanisms of action, still remain. This overview attempts to focus on some of these questions. Four aspects of modification of chemotherapy by nitroimidazoles are reviewed and discussed. These address the importance in chemopotentiation of i) hypoxia, ii) alterations in DNA damage and/or repair, iii) depletion of intracellular sulfhydryls and iv) modification of drug pharmacokinetics. It is concluded that: i) even though chemopotentiation can occur at intermediate oxygen levels, hypoxia ultimately plays a pivotal role, ii) no single unifying mechanism for chemopotentiation exists; alterations in drug pharmacokinetics, cellular SH levels and DNA damage/repair all are involved, the relative importance of each factor is dependent on the particular drug-sensitizer combination, iii) it is important to continue the evaluation of chemopotentiation under conditions mimicking clinically achievable sensitizer pharmacokinetics and iv) further investigations into more effective utilization of chemopotentiation are warranted.

Relationship between radiobiological hypoxia in tumors and electrode measurements of tumor oxygenation

PURPOSEnTo determine whether electrode measurements of tumor oxygenation, made in a variety of murine tumor models, correlate with estimates of radiobiological hypoxia in the same tumor systems.nnnMETHODS AND MATERIALSnThe tumor models used were a C3H mammary carcinoma grown in the feet of CDF1 mice; the SCCVII, KHT and RIF-1 tumors grown in the feet or flanks of C3H/Km mice; and the CaNT and SaF tumors grown on the backs of CBA mice. All treatments were performed when tumors were about 200 mm3 in size. Radiobiological hypoxic fractions were determined using either a paired survival curve assay, with survival measured 0-24 h after irradiation, or using a clamped tumor control assay, with percent local tumor control estimated 90 days after treatment. Measurements of tumor oxygen partial pressure (pO2) distributions were performed using Eppendorf oxygen electrodes.nnnRESULTSnThe hypoxic fractions determined from the radiation response data were about 1% in RIF-1 and SCCVII, 12% in C3H and KHT, 28% in CaNT and up to 38% in SaF tumors. When this data was compared with the tumor oxygenation measurements it was found that as hypoxic fraction increased the mean, median, and the percentage of pO2 values < or = 5 mmHg showed a trend towards poorer oxygenation status. However, none of these pO2 changes were significantly correlated with hypoxia. Moreover, the pO2 values < or = 2.5 mmHg indicated an improvement in oxygen status with increasing hypoxic fraction.nnnCONCLUSIONnElectrode measurements of tumor oxygenation alone may, therefore, not be a good indicator of tumor hypoxia across different tumor cell lines.

Effects of oxygenation and ph on tumor cell response to alkylating chemotherapy

In the present investigations we evaluated the consequence of changing the cellular microenvironment on the treatment efficacy of the alkylating chemotherapeutic agent melphalan. Human A549 adenocarcinoma and mouse KHT/iv sarcoma cells were treated with melphalan under aerobic or hypoxic conditions at pH 6.6 or 7.4. Both low oxygenation and acidic pH individually were found to increase tumor cell killing by this chemotherapeutic agent. However, the magnitude of the enhanced toxic effect was greatest when hypoxic conditions and acidic pH were combined during treatment. For example, A549 cells treated with melphalan under hypoxic conditions at pH 6.6 were approximately 3 times more sensitive to this anticancer drug than were cells exposed in air at pH 7.4. Conditions of low oxygen and pH also increased the chemosensitization potential of the nitroimidazole misonidazole (MISO) when combined with this chemotherapeutic agent. Thus, when KHT/iv cells were treated with the combination of melphalan plus MISO, the resulting enhancement ratio increased from 1.8 to 2.5, when the pH maintained during the treatment was changed from physiologic (7.4) to acidic (6.6).

Depletion of tumour versus normal tissue glutathione by buthionine sulfoximine

We have investigated in detail the effects of buthionine sulfoximine (BSO), a selective glutathione (GSH) depleting agent, on the GSH contents of a number of normal tissues and three experimental tumours in mice. Significant variations in the rate and degree of GSH depletion and recovery were observed among the normal tissues. Following a dose of 2.5 mmol kg-1 BSO, GSH nadirs were reached by approximately 5 h for the liver and kidney, 8 h for the lung and bone marrow, 12 h for red blood cells (RBCs) and by 24 h for the heart. The degree of depletion was greatest for the kidney (80%), liver (74%) and bone marrow (83%), intermediate for the heart (54%) and lung (40%), and least for RBCs (13%). Recovery of GSH content was fastest for the liver followed in descending order by the kidney, the lung, the bone marrow, RBCs and the heart. In contrast, the rate and extent of GSH depletion and recovery showed considerably less variation among the 3 murine tumours. In the tumours GSH nadirs were reached by 10-12 h. The extent of depletion was about 55-65%. Recovery of GSH levels in the tumours required 48 h or more, a longer period than required by the liver, kidney and lung but shorter than that needed for the bone marrow, heart and RBCs. Attempts to preferentially deplete tumour GSH by exploiting the differences in recovery rates between normal tissues and tumours were only partially successful. Multiple BSO dosing at 16 h intervals allowed the liver to recover between doses, but the recovery in the kidney, lung and bone marrow was only partial and no recovery was seen in the heart. Finally, dose-depletion relationship investigations showed that, with the exception of the lungs, GSH depletion could be achieved in tumours with doses of BSO lower than those required for normal tissues.

Early and late pulmonary toxicity in mice evaluated 180 and 420 days following localized lung irradiation

The lungs of 2 to 3-month-old female C3H/HeJ and 6-month-old male LAF1 mice were locally irradiated with graded doses of radiation ranging from 900 to 1800 rad. The patterns of animal details for times up to 60 weeks after irradiation were recorded. Over this period mice in all the irradiated groups continued to die. However, the onset of animal deaths was dose dependent such that lethality occurred earlier after the higher radiation doses. Whereas mice receiving large radiation doses started to die by 75-100 days post-treatment, few or no animal deaths occurred in those groups receiving the lowest doses until 200-300 days after irradiation. At 180 and 420 days postirradiation animal lethality dose-response curves were constructed; these were found to be similar in shape (sigmoid), but at the later time the calculated LD/sub 50/ values were approx.300-400 rad lower than those determined using the 180-day post-treatment end point. Histological examination of the irradiated lungs indicated that the animal deaths occurring by 180 days were associated with radiation pneumonitis while the late deaths were associated with pulmonary fibrosis. The addition of the radiation sensitizer misonidazole prior to lung irradiation did not significantly enhance pulmonary toxicity at either end point.

Reducing Acute and Chronic Hypoxia in Tumours by Combining Nicotinamide with Carbogen Breathing

The ability of nicotinamide and carbogen breathing to improve the radiation response of a C3H mammary carcinoma by reducing both acute and chronic hypoxia was investigated. Using a tumour growth delay assay the response of 200 mm3 foot tumours to local irradiation was found to be increased by either injecting nicotinamide (100-1,000 mg/kg) 20 min prior to irradiation, or by allowing mice to breathe carbogen for 10 min before and during the radiation treatment. The greatest radiosensitization occurred when nicotinamide and carbogen were combined. With a histological fluorescent staining technique nicotinamide was shown to prevent transient stoppages in microregional blood flow, and also appeared to improve tumour oxygenation as measured with an Eppendorf oxygen electrode, both effects being consistent with its ability to decrease perfusion limited acute hypoxia. Carbogen had no effect on vessel closure, but it significantly improved tumour oxygenation, which was indicative of it reducing diffusion limited chronic hypoxia.

Surfactant release as an early measure of radiation pneumonitis

The immediate release of surfactant into lung alveoli following irradiation has been studied as a potential indicator for the later development of radiation pneumonitis. Utilizing single dose radiation exposure to the whole thorax in male LAF1/J mice, steep dose response curves for lavaged alveolar surfactant were identified at 7 and 28 days after exposure. Seven days after irradiation there was no elevation with doses up to and including 12 Gy; above this dose a detectable increase occurred. At 28 days the surfactant recovered by lavage was elevated compared to the levels seen at day 7 for all doses; doses greater than 12 Gy produced surfactant values significantly greater than those found in mice treated with 12 Gy or less. The radiation pulmonary lethality dose response curve assessed four months later indicated an LD50 value of approximately 13 Gy. The early biochemical effect and the later radiation pneumonitis lethalities therefore closely coincided. The evidence strongly indicates that alveolar surfactant release uncovered hours to days after radiation exposure may be an early biochemical marker that predicts for subsequent pneumonitis radiation injury.

Further evaluation of nicotinamide and carbogen as a strategy to reoxygenate hypoxic cells in vivo: importance of nicotinamide dose and pre-irradiation breathing time

The combination of nicotinamide and carbogen breathing is awaiting clinical evaluation as a strategy to overcome tumour hypoxia and thus enhance radiation response. We have continued our evaluation of this approach in the murine SCCVII tumour with the aim of determining the importance of nicotinamide dose and the pre-irradiation breathing time (PIBT) for carbogen. For carbogen breathing alone maximal enhancement of radiation response was observed with PIBTs of between 5 and 30 min. When nicotinamide (1,000 mg kg-1 IP) was administered 60 min prior to irradiation little or no variation in radiation response was observed for all the PIBTs examined (5-90 min). Indeed at all PIBTs the cell survival obtained for the carbogen nicotinamide and radiation combination was indistinguishable from that expected for a fully aerobic response. For PIBTs of 15 and 60 min we examined the influence of nicotinamide doses between 50 and 1,000 mg kg-1. Significant radiosensitizing effects were observed for all nicotinamide doses tested above 50 mg kg-1. Moreover for doses of 250 mg kg-1 and above the cell survival data was consistent with that expected for a fully aerobic response. No additional benefit accrued from raising the nicotinamide dose above 250 mg kg-1. These results indicate that significant radiosensitization may be expected even with clinically achievable nicotinamide doses when it is combined with carbogen breathing. Furthermore, the use of nicotinamide may reduce the critical importance of PIBT on the radiosensitization observed with carbogen.

Changes in cellular glutathione content during adriamycin treatment in human ovarian cancer--a possible indicator of chemosensitivity.

Patients with ovarian cancer often respond well to combination chemotherapy initially but the majority eventually relapse when, with further treatment, the initially successful regimen proves ineffectual. The cause of such failures frequently has been attributed to the development of drug resistance. Although the mechanisms of acquired resistance in situ are still poorly understood, studies in vitro have shown that cells selected for resistance to one drug often exhibit cross-resistance to other seemingly unrelated agents, suggesting a somewhat generalised mechanism of resistance. We have studied the role of glutathione (GSH) and drug transport in determining the sensitivity to adriamycin (ADR) of a panel of human ovarian cell lines established directly from biopsies of patients with diverse treatment histories. These cell lines exhibited inherent differences in sensitivity to ADR by a dose factor of up to 3; a difference that was considerably less than what has been reported when cells were selected for drug resistance in vitro. The differences in drug sensitivity reported here among the various cell lines appeared to be unrelated to drug transport, in terms of both influx and efflux. Moreover, although these cell lines have a wide range of GSH content, there was only a poor correlation between drug sensitivity and cellular GSH content per se. However, when exposed to a clinically relevant dose of ADR, the GSH content of cell lines that were sensitive decreased, whereas that of cell lines that were resistant increased. To take these time-dependent changes in GSH into consideration, the area under the GSH content versus time curve (AUC), with and without ADR treatment, was calculated for each cell line. When this latter factor was included in the analysis, greatly improved correlations were found between GSH kinetic parameters and responses to ADR. In particular, ADR resistance was found to be closely correlated with the positive changes in absolute GSH AUC following ADR treatment (r = 0.92; P less than 0.01). Using 35S-labelled cysteine and methionine as tracers, it was found that the essential difference between the resistant and sensitive lines was that the resistant lines had higher steady-state rates of GSH synthesis than the sensitive lines. These results demonstrate that changes in cellular GSH concentration during treatment may be an important indicator of tumour cell response to ADR.