Radiosensitization of tumor-targeted radioimmunotherapy with prolonged topotecan infusion in human breast cancer xenografts.

Clinical radioimmunotherapy (RIT) of solid tumors holds great promise, but as yet has been unable to deliver tumoricidal radiation doses without unacceptable toxicity. Our experimental approach aims to potentiate the therapeutic action of radioimmunoconjugates at the tumor site and thus improve the efficacy of RIT by combination with other treatment modalities. The topoisomerase I inhibitors are a unique class of chemotherapeutic agents that interfere with DNA breakage-reunion by inhibiting the action of topoisomerase I. Preclinical studies suggest that prolonged infusion of topoisomerase I inhibitors enhances cell toxicity due to ionizing radiation. We evaluated the efficacy of combined treatment with continuous administration of topotecan and 90Y-MX-DPTA BrE3 monoclonal antibody (which recognizes an epitope of breast epithelial mucin expressed in most breast cancers) on human mammary carcinoma xenografts in nude mice. Topotecan or 90Y-BrE3 treatment alone delayed overall tumor growth rate transiently but did not affect survival. The combination of RIT with topotecan substantially reduced growth of relatively large established tumors and caused complete tumor regressions and prolonged tumor-free survival in a substantial proportion of treated animals. In vitro studies demonstrated an increase in apoptotic rate and a decrease in cell proliferation of tumor cell lines treated with this combination. We combined the radiosensitization property of topotecan and the specificity of systemic RIT to establish a novel therapy for solid tumors in an experimental tumor xenograft model.

Influence of lactoferrin feeding and injection against systemic staphylococcal infections in mice.

Human and bovine lactoferrins (Lfs) and bovine lactoferrin hydrolysate (LH) were assessed in vitro and in vivo for their antibacterial effects on Staphylococcus aureus. Lactoferrins showed weak in vitro antibacterial activity while Fe-saturated Lfs and LH showed no activity. Lactoferrin-treated mice (1 mg, i.v.) when injected i.v. with 10(6) staphylococci, showed 30-50% reduction in kidney infections, and viable bacterial counts in the kidneys decreased 5-12-fold. The inhibitory effect was dose-dependent up to 1 mg Lf. Lactoferrins were effective when given 1 day prior to the bacterial challenge, after which there was no significant effect even at doses up to 5 mg. Apo- and Fe-saturated forms of human and bovine Lfs were all equally effective, while LH was not protective. Human and bovine Lfs with different degrees of iron saturation (9-97%) were found to be equipotent. Feeding mice with 2% bLf in drinking water also reduced the kidney infections by 40-60%, and viable bacterial counts, 5-12-fold. The results suggest a potential for the use of Lfs as natural antibacterial proteins for preventing bacterial infections.

Induction of lactoferrin expression in murine ES cells by retinoic acid and estrogen.

Lactoferrin is an iron-binding glycoprotein present in high concentrations in milk and exocrine fluids such as bile and tears. Many functions have been attributed to lactoferrin, including antimicrobial and antiviral activities, immunomodulation, and cell growth regulation. Lactoferrin expression is controlled by different regulators, including retinoic acid and estrogen. However, the expression pattern of lactoferrin in mammalian early development has not yet been reported. Murine embryonic stem cells are pluripotent cells that can contribute to all tissues and were used for this study. We show here that while no lactoferrin protein or mRNA was detected in untreated murine embryonic stem cells, retinoic acid and estrogen can induce high levels lactoferrin expression in these cells. Expression, demonstrated by reverse transcription-polymerase chain reaction, Western blot, immunofluorescence, and ELISA assay, was dose and time dependent. Our study provides an in vitro model for examining lactoferrin expression in early development and differentiation.

Biologic activity of interleukin 1 (IL-1) alpha in patients with refractory malignancies.

Interleukin 1 alpha (IL-1 alpha) is a cytokine with pleiotropic effects, including cytotoxic-cytostatic activity against some tumor cell lines. We have conducted a phase I study of recombinant human IL-1 alpha (rhIL-1 alpha) in 17 patients with refractory malignancies to examine its toxicity and biologic activity. rhIL-1 alpha was given as a 2-h IV infusion daily for 5 days at five dose levels (0.08, 0.2, 0.8, 2.0, and 5.0 micrograms/m2). Seventeen patients with malignancies were treated, with no objective tumor responses noted. Common toxicities included: fever (100%), rigors and/or chills (96%), myalgia (54%), and headache (48%). Three patients developed grade III hypotension. The maximum tolerated dose was 2.0 micrograms/m2. rhIL-1 alpha induced a significant increase in absolute neutrophil count over baseline (p < 0.05), a delayed but significant increase in platelet count over baseline (p < 0.05), and there was a marked increase in the number of progenitors [colony-forming units (CFU)-G, CFU-M, CFU-GM, CFU-GEMM and burst-forming units (BFU-E)] observed in the peripheral blood. Nine of 12 evaluable patients showed an increase in bone marrow cellularity or myeloid:erthyroid ratio. Immunophenotyping did not demonstrate an increase in peripheral blood or bone marrow CD34+ cells. Interferon-gamma-mediated monocyte cytotoxicity (MCCTX) was significantly enhanced from baseline (p < 0.001), although an increase in direct MCCTX did not reach statistical significance. In summary, rhIL-1 alpha administration is well tolerated at a dose of 2.0 micrograms/m2 with fever, rigors, myalgia, and headache being the most frequent toxicities. Although there were no objective tumor responses, we have demonstrated significant biologic activity with increased neutrophil and platelet counts, increased peripheral blood progenitor cells, and enhanced interferon-gamma-mediated MCCTX.

Initial clinical evaluation of radiolabeled MX-DTPA humanized BrE-3 antibody in patients with advanced breast cancer.

To evaluate radiometal-labeled humanized BrE-3 (huBrE-3) monoclonal antibody as a radioimmunolocalization and therapeutic agent in breast cancer patients, tumor localization, pharmacokinetics, radiation dosimetry, and immunogenicity of (111)In-labeled combined 1-p-isothiocyanatobenzyl 3-methyl- and 1-p-isothiocyanatobenzyl 4-methyldiethylenetriamine pentaacetic acid (MX-DTPA) huBrE-3 were studied. Seven women with BrE-3 antigen-positive, metastatic breast carcinoma underwent (111)In huBrE-3 infusion (5 mCi; 50 mg), followed by serial gamma camera imaging and plasma sampling. Region of interest analysis of images was used to make radiation absorbed dose estimates for (111)In huBrE-3. Data were extrapolated to 90Y huBrE-3. Human anti-human antibody (HAHA) response was measured in serum samples obtained up to 3 months after infusion. Patients tolerated infusions well. Seventy-six percent of 105 known sites of disease were identified on planar and single-photon emission computed tomography scans. For six of seven patients, a biexponential model fit the plasma time-activity curve best with an average T1/2alpha=10.6+/-8.5 (SD) h and average T1/2beta=114.2+/-39.2 h. Radiation absorbed dose estimates for (111)In huBrE-3 for whole body averaged 0.53+/-.08 rads/mCi. Dose estimates for 90Y huBrE-3 for marrow averaged 8.4+/-11.9 rads/mCi, and for tumors, 70+/-31.5 rads/mCi. Liver radioactivity uptake averaged 19.7+/-8.8% injected dose at 24 h after infusion, translating into an average radiation absorbed dose 21.1+/-12 rads/90Y mCi administered. Only one of seven patients demonstrated a low level of HAHA response. Although the plasma half-lives are longer and marrow dose higher for radiolabeled huBrE-3 compared with the murine construct, the excellent tumor localization, good tumor dosimetry, and low immunogenicity support the use of 90Y-huBrE-3 antibody for radioimmunotherapy of breast cancer.

The effect of continuous infusion IL-1 alpha on carboplatin-induced thrombocytopenia and anti-tumor activity in RIF-1 tumor bearing mice.

Interleukin-1 alpha (IL-1 alpha) has potent effects on hematopoiesis and can significantly enhance the anti-tumor activity of cytotoxic drugs. Studies were undertaken here to determine whether IL-1 alpha, administered by continuous infusion, could prevent carboplatin (CBDCA)-mediated thrombocytopenia and enhance CBDCA-mediated anti-tumor effects. RIF-1 tumor bearing mice were treated with CBDCA and IL-1 alpha either by a single bolus injection or by continuous infusion through the use of ALZET pumps. The duration and extent of CBDCA-induced thrombocytopenia in tumor-bearing mice was diminished when IL-1 alpha was administered continuously as compared to CBDCA alone or CBDCA plus a single bolus injection of IL-1 alpha. In addition, IL-1 alpha induced potentiation of CBDCA anti-tumor activity in vivo was significantly increased when IL-1 alpha was administered by continuous infusion. These results demonstrate the potential efficacy of IL-1 alpha by continuous infusion.

Effect of high-dose, fractionated local irradiation on MNU-induced carcinogenesis in the rat mammary gland.

The effect on chemical carcinogenesis in the mammary gland of high-dose fractionated, local irradiation, as is used in the treatment of human breast cancer, was examined in a rat model of this disease process. For this purpose, a highly reproducible method was employed for administering a therapeutic dose and fractionation schedule via an anterior portal to a single mammary gland chain of rats in a manner that minimized whole body irradiation. This approach offers significant advantages over whole body irradiation techniques previously used for investigations of radiation-mediated effects on the carcinogenic process. Among the advantages are that higher doses of radiation can be administered to the target tissue with minimal side effects and that the contralateral mammary gland chain can serve as a 'within animal control'. When this approach was used to study the effect of high-dose fractionated radiation on the risk of development of mammary cancer in rats given 1-methyl-1-nitrosourea prior to radiation, an enhanced tumorigenic response was observed that greatly exceeded the response resulting from either radiation or carcinogen administered alone. This result was unanticipated, based on data from animal studies of the effects of whole body irradiation on mammary tumor development and the outcome of clinical series. Possible reasons for the discrepancy are presented.

Sequence specificity and transcriptional activation in the binding of lactoferrin to DNA.

Lactoferrin, an iron-binding glycoprotein found in high concentrations in human milk and other epithelial secretions and in the secondary (specific) granules of neutrophils, is thought to be responsible for primary defence against microbial infection, mainly as a result of lactoferrin sequestration of iron required for microbial growth. Many other functions have been attributed to lactoferrin, including immunomodulation and cell growth regulation (reviewed in ref. 4). Some of these functions appear to be at least in part independent of the iron-binding activity of lactoferrin. It also has been consistently observed that lactoferrin interacts avidly with nucleic acids. Lactoferrin enhancement of the activity of natural killer and lymphokine-activated killer cells in vitro is inhibited by RNA and DNA. Lactoferrin taken up by K562 human myelogenous leukaemia cells appears in the nucleus where it is bound to DNA. We report here that binding of lactoferrin to DNA occurs under stringent conditions with distinct sequence specificity, and that interaction between lactoferrin and these sequences intracellularly leads to transcriptional activation.

Potentiation by interleukin 1 alpha of cisplatin and carboplatin antitumor activity: schedule-dependent and pharmacokinetic effects in the RIF-1 tumor model.

We have previously demonstrated that the cytokine interleukin 1 alpha (IL-1 alpha) significantly potentiates the antitumor activity of a variety of chemotherapeutic agents, including cisplatin (cDDP). In studies described here, we examined the potential of combining IL-1 alpha and the platinum analogue carboplatin (CBDCA) and compared the schedule-dependent and pharmacokinetic effects for IL-1 alpha combinations with cDDP and CBDCA. RIF-1 tumor-bearing mice (C3H/HeJ) received i.p. injections of varying doses of CBDCA, alone or concurrently with IL-1 alpha (48 or 480 micrograms/kg). Clonogenic cell kill and tumor regrowth delay were significantly increased when CBDCA was combined with IL-1 alpha, at both doses, compared to either CBDCA or IL-1 alpha alone (P < 0.001 and P < 0.01, respectively). Although pretreatment with IL-1 receptor antagonist blocked the acute tumor hemorrhagic response induced by IL-1 alpha alone, IL-1 receptor antagonist only partially blocked IL-1 alpha enhancement of CBDCA or cDDP-mediated tumor cell kill. The IL-1 alpha enhancement of CBDCA-mediated tumor cell kill was highly schedule dependent, with maximum antitumor activity observed when IL-1 alpha was administered 4-12 h before CBDCA. In contrast, administration of IL-1 alpha from 24 h before or as late as 6 h after cDDP resulted in the same antitumor activity as simultaneous administration of cDDP and IL-1 alpha. Tumor and normal tissue platinum content were significantly increased by IL-1 alpha in animals treated with CBDCA (P < 0.01) but not in those treated with cDDP. The observed differences between cDDP and CBDCA may be explained by their known differential rates of clearance and protein binding affinities and are compatible with an induced alteration in CBDCA pharmacokinetics.

Human lactoferrin inhibits growth of solid tumors and development of experimental metastases in mice.

The antitumor effects of the multifunctional iron-binding glycoprotein, lactoferrin (Lf), were investigated. Lf inhibited growth in mice of transplantable solid tumors induced by v-ras transformed fibroblasts and a methylcholanthrene-induced fibrosarcoma. Lf also substantially reduced lung colonization (experimental metastasis) by B16-F10 melanoma cells in syngeneic mice. Iron-saturated and apo-Lf exhibited comparable levels of tumor inhibition and antimetastatic activity. Transferrin, a related iron-binding protein, had no effect on lung colonization. In the B16-F10 system, elimination of natural killer cell activity by pretreatment of mice with anti-asialo GM1 antibody abrogated the effects of Lf, whereas inhibition of macrophage function with silica did not. The results demonstrate a novel activity for Lf and suggest a potentially important role for this molecule in the primary defense against tumorigenesis.

Iron-induced conformational change in human lactoferrin: demonstration by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analysis of effects of iron binding to the N and C lobes of the molecule.

Analysis of Fe-saturated- and apo-lactoferrin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) without heating the samples prior to application revealed a substantial difference in mobility. The mobility shift was fully reversible on repetitive removal and readdition of Fe. Binding of a single Fe to the N-lobe binding site was sufficient to cause the gel shift; binding of a second Fe to the C-lobe site did not further alter mobility. Removal of Fe from the N lobe of Fe2 lactoferrin did not restore mobility to the position of apolactoferrin. No change in mobility with Fe binding was detected in N and C lobes isolated from intact lactoferrin by controlled trypsin digestion. The data indicate that a conformational change induced by Fe binding to a single site on lactoferrin is detectable by SDS-PAGE and that this change requires an intact molecule, possibly due to the need for interactions between the two homologous lobes of the molecule.

Analysis of the antimetastatic effects of synthetic muramyl tripeptide (CGP 19835A) encapsulated in liposomes in combination with other immunomodulatory agents and chemotherapeutic drugs.

The synthetic molecule muramyl tripeptide (CGP 19835A) encapsulated in liposomes is effective in increasing the survival of mice with spontaneous experimental lung metastases induced by the RENCA renal adenocarcinoma and B16 melanoma tumor models. The present study was aimed at extending the effects of CGP 19835A to another highly metastatic carcinoma model and at evaluating the efficacy of combination therapy with standard cytotoxic agents and other immunomodulators. C57BL/6 mice received whole tumor implants of PancO2, a spontaneously metastasizing pancreatic adenocarcinoma, subcutaneously in the hind leg. Therapeutic effects were measured by increased survival which is a direct function of the growth of spontaneous lung metastases in this system. No therapeutic efficacy was observed with CGP 19835A alone or in combination with any of a series of cytotoxic or biological agents, including cis-platinurn (cis-Pt), mitomycin C (MMC), adriamycin (ADR), cyclophosphamide (CP), interferon gamma (IFN gamma), and interleukin 2 (IL-2). In accord with previous studies, when the B16-F10 melanoma was used as an experimental metastatic tumor model, CGP 19835A, alone and in combination with CP, significantly reduced the number of pulmonary metastases. Cis-Pt, however, partially negated the effects of CGP 19835A when a combination of the two agents was used. The results indicate that CGP 19835A is an effective therapeutic agent in some models of spontaneous or experimental lung metastases, but not others, and that the effects of CGP 19835A are not enhanced by the accompanying cytotoxic drugs tested here.

Iron binding to human lactoferrin alters reactivity of the protein with plant lectins.

Binding of Fe by human apolactoferrin results in altered reactivity of the glycoprotein with plant lectins. Reaction with wheat germ agglutinin (WGA) and peanut agglutinin (PNA) was abolished with Fe binding. Reaction with the lectins from Datura stramonium (DSA) and Aleuria aurantia (AAA) was significantly reduced but not fully abolished on Fe binding, while reaction with the Artocarpus integrifolia lectin (Jacalin) and Sambucus nigrabark (SNA) was not changed at all. Loss of WGA reactivity occurred when only one of two Fe binding sites on the molecule was saturated. The results demonstrate conformational changes that are associated with high-avidity binding of Fe by lactoferrin.

Radioimmunolocalization of metastatic breast carcinoma using indium-111-methyl benzyl DTPA BrE-3 monoclonal antibody: phase I study.

Pharmacokinetics of radiolabeled BrE3 monoclonal antibody (Mab), reactive against a breast mucin epitope, were assessed in 15 patients with advanced breast cancer. Patients received 5 mCi (185 MBq) of 111In-methyl benzyl isothiocyanate DTPA (MX-DTPA) conjugated BrE-3 Mab intravenously with total antibody doses of 10, 50 or 100 mg. Serial quantitative imaging, blood and urine clearance were obtained to measure pharmacokinetics, assess tumor localization and estimate radiation dose. Organ function was followed to determine toxicity. Mild allergic reactions occurred in four patients. Eighty-six percent of 70 known lesions and 5 unsuspected lesions were detected by antibody imaging. Biexponential modeling of radiolabeled antibody in serum showed a T1/2 alpha = 9.5 +/- 2.7 hr and T1/2 beta = 56 +/- 25.4 hr. Total urinary excretion averaged 35.5% +/- 19.3% injected dose (ID) by Day 8. Quantitative imaging showed that 0.02-2.56% ID localized in tumors. Extrapolating dosimetry from 111In-MX-DTPA-BrE-3 to 90Y-MX-DTPA-BrE-3, we estimate therapeutic radiation doses could be delivered to some tumors with tolerable toxicity.

Effect of conjoint administration of tamoxifen and high-dose radiation on the development of mammary carcinoma.

PURPOSE: Tamoxifen is currently advocated for post-menopausal breast cancer patients receiving definitive irradiation after limited surgery. The purpose of this study was to assess in an experimental model for breast cancer whether the efficacy of irradiation is altered by conjoint administration of tamoxifen. To this end, rats with small tumors induced by 1-methyl-1-nitrosourea (MNU) were treated with tamoxifen, radiation, or a combination of the two modalities. METHODS AND MATERIALS: Female Sprague Dawley rats were injected i.p. with 50 mg MNU/kg body weight at 50 days of age. At 64 days post carcinogen, the majority of the rats had at least one palpable mammary tumor. At that time radiation with or without tamoxifen treatment was initiated and given 5 days per week for 5 weeks. Radiation dose was 4500 cGy delivered as 25, 180 cGy fractions. Tamoxifen, 500 mg/kg body weight, was administered subcutaneously each day during the irradiation interval. The study was terminated 28 weeks after carcinogen treatment. RESULTS: High dose radiation alone induced a reduction in the size of existing tumors, but resulted in a significant increase in the number of tumors that were detected. Treatment with tamoxifen alone also caused a reduction in tumor volume, but had no effect on final incidence or number of mammary tumors. Combined modality treatment resulted in a significant reduction in the volume of existing tumors and suppressed the enhanced occurrence of additional tumors observed when only radiation alone was administered. CONCLUSION: The findings of this study indicate that in the context of fractionated, high dose radiation treatment of established mammary cancers, tamoxifen may reduce the likelihood of subsequent tumor development and by so doing prove a helpful simultaneous conjoint adjuvant treatment to post-operative irradiation.

Synergistic enhancement by interleukin-1 alpha of cisplatin-mediated antitumor activity in RIF-1 tumor-bearing C3H/HeJ mice.

Administration of interleukin-1 alpha (IL-1 alpha) plus certain cytotoxic drugs causes substantially greater clonogenic tumor-cell kill and tumor-regrowth delay than does treatment with either agent alone. IL-1 alpha itself has little effect on tumor growth despite its ability to induce acute hemorrhagic necrosis, restrict tumor blood flow, and cause microvascular injury in a variety of murine model systems. To investigate further IL-1 alpha's ability to enhance the antitumor activity of cytotoxic drugs, we initiated studies to examine the effect of IL-1 alpha on cisplatin (cDDP)-mediated cytotoxicity using the RIF-1 tumor system. The antitumor activity of IL-1 alpha and cDDP was quantitated through standard clonogenic tumor-cell survival assays, a tumor hemorrhagic necrosis assay and tumor-regrowth delay studies, with the interaction between IL-1 alpha and cDDP being analyzed through median dose-effect. In vitro, IL-1 alpha had no enhancing effect on the cDDP-mediated tumor-cell kill. For examination of the in vivo efficacy of this regimen, RIF-1 tumor-bearing C3H/HeJ mice (14 days postimplantation) were treated concurrently with single i.p. injections of IL-1 alpha and/or cDDP at various doses. The increased clonogenic tumor-cell kill obtained with IL-1 alpha/cDDP was dose-dependent, with significant enhancement by IL-1 alpha being observed (P < 0.001), even at the lowest doses tested (2 mg/kg and 6 micrograms/kg for cDDP and IL-1 alpha, respectively), but it did not correlate with an increase in tumor hemorrhage. Using median dose-effect analysis, this interaction was determined to be strongly synergistic. When treated animals were monitored for long-term antitumor effects, combinations with IL-1 alpha significantly increased the tumor-regrowth delay and decreased the fractional tumor volume (P < 0.001). These results demonstrate that IL-1 alpha synergistically enhances cDDP mediated in vivo antitumor activity and suggest that the combination of IL-1 alpha and cDDP may have potential therapeutic application in the design of effective treatment modalities for cancer.

Differential effect in vitro of tumor necrosis factor-alpha (TNF) on normal and virus-infected erythroid progenitors from Friend virus (FVA)-infected mice.

In vivo administration of tumor necrosis factor-alpha (TNF) suppresses both normal and Friend virus (FVA)-infected erythroid progenitor cells (CFU-E). To examine the mechanism of erythroid suppression by TNF, we examined TNF's direct effect on normal and virus-infected cells in vitro. Productively infected fibroblast cell lines, fresh acute virus-infected spleen cells, and virus-infected CFU-E were sensitive, whereas uninfected CFU-E were resistant to TNF cytotoxicity in vitro. When FVA-infected erythroblasts were depleted from the spleen cell population in vitro with antivirus antibodies, TNF suppression of the remaining (uninfected) cells was abrogated. In contrast, both normal and virus-infected macrophage progenitor cells and immature erythroid progenitor cells were equally sensitive to TNF cytotoxicity in vitro. Normal erythroblasts had significantly fewer TNF receptors than FVA-infected erythroblasts, which also were morphologically less mature. These results suggest that TNF can differentially suppress late-stage virus-infected erythroid progenitors in vitro.