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Donald J Buchsbaum

Publications and source records attributed to Donald J Buchsbaum.

36 records · Page 2Linked to original sources
PubMed2003

Antitumor efficacy of TRA-8 anti-DR5 monoclonal antibody alone or in combination with chemotherapy and/or radiation therapy in a human breast cancer model.

PURPOSE: A monoclonal antibody (TRA-8) has been developed that binds to death receptor 5 (DR5), one of two death receptors bound by tumor necrosis factor-related apoptosis-inducing ligand. The purpose of this study was to evaluate in vitro the binding and cytotoxicity of TRA-8 to human breast cancer cell lines. The antitumor efficacy of TRA-8 was evaluated in a xenograft human breast cancer murine model, as a single agent and in combination with chemotherapy or radiation therapy. Anti: The binding of TRA-8 to a panel of nine human breast cancer cell lines was evaluated by indirect immunofluorescence and flow cytometry. Cytotoxicity of TRA-8 alone and in the presence of Adriamycin or paclitaxel was measured in vitro using the ATP-lite assay. Antitumor efficacy was determined by treatment of nude mice bearing well-established s.c. DR5-positive 2LMP human breast cancer xenografts with TRA-8 alone or in combination with Adriamycin or paclitaxel. Tumor size and regression rates were determined. In addition, a study was carried out with TRA-8 and Adriamycin in combination with 3 Gy (60)Co irradiation of 2LMP xenografts on days 9 and 17. RESULTS: All nine human breast cancer cell lines expressed DR5 with TRA-8 reactivity varying from strongly to weakly positive. Four cell lines were sensitive to TRA-8 cytotoxicity with IC(50) of 17-299 ng/ml, whereas other cell lines had weak cytotoxicity or were resistant. In vivo studies demonstrated significant inhibition of growth of 2LMP xenografts by TRA-8 treatment alone. The combination of TRA-8 + Adriamycin or paclitaxel produced significant inhibition of tumor growth as compared with controls or either agent alone. An aggregate analysis of all 166 animals studied demonstrated that TRA-8 alone or in combination with Adriamycin, paclitaxel, or radiation produced a significant increase in tumor doubling time compared with any modality alone with mean doubling time in days of 12 (untreated), 14 (radiation), 17 (Adriamycin), 25 (paclitaxel), 39 (Adriamycin + radiation), 47 (TRA-8), 65 (TRA-8 + radiation), 71 (TRA-8 + paclitaxel), 81 (TRA-8 + Adriamycin), and >140 (TRA-8 + Adriamycin and radiation). Complete tumor regressions occurred in 1 of 42 untreated animals, 1 of 54 animals receiving chemotherapy and/or radiation, and 28 of 68 animals receiving TRA-8 alone or TRA-8 combination regimens. Fourteen of those 28 complete regressions did not relapse over periods of follow-up between 99 and 171 days, with a mean of 146 +/- 24 days. CONCLUSIONS: The TRA-8 anti-DR5 antibody alone or in combination with chemotherapy and/or radiation has striking antitumor efficacy in breast cancer xenograft models. Additional studies with other tumor types and chemotherapy agents are warranted. These studies support the generation of a humanized TRA-8 for introduction into early clinical trials.

Animals↗
PubMed2003

Differential responses by pancreatic carcinoma cell lines to prolonged exposure to Erbitux (IMC-C225) anti-EGFR antibody.

BACKGROUND: Pancreatic cancer remains a devastating disease, with 95% of all patients diagnosed with the disease dying within 2 years. The combined therapy using Erbitux, gemcitabine, and radiation caused complete tumor regression using a nude mouse model inoculated with pancreatic MiaPaCa-2 cells but only a delay in tumor growth with BxPC-3. We investigated the effect of prolonged Erbitux treatment to the sensitivity to gemcitabine and/or radiation and the epidermal growth factor receptor (EGFR) signal transduction pathway. METHODS: MiaPaCa-2 and BxPC-3 cells were cultured with or without Erbitux for 6 weeks. Cells were then treated with gemcitabine and/or radiation, harvested 48 h after treatment, and counted. Differences in EGFR expression after exposure to Erbitux were analyzed by FACS. Internalization rates of EGFR induced by Erbitux on these cell lines were determined using 125I-EGF binding assay after removal of Erbitux by acidic wash. Cell lysates were harvested after cells were stimulated with EGF, FGF, or IGF-1 respectively, and EGFR was immunoprecipitated using Erbitux. Samples were separated using SDS-PAGE and transferred to PVDF membrane. The membranes were probed with antibody against human growth factor receptor binding protein (Grb2) to detect the association of this Ras-MAPK upstream adaptor protein to EGFR. Cell lysates were also separated with SDS-PAGE and probed with rabbit anti-human PARP after samples were transferred to PVDF membrane. Expression of BAX and Bcl-(XL) were probed in the cells treated with or without Erbitux. RESULTS: Proliferation assays indicated that prolonged exposure to Erbitux increased the sensitivities of MiaPaCa-2 to gemcitabine and radiation therapy (41 +/- 16% vs 52 +/- 9% for gemcitation, 28 +/- 9 vs 39 +/- 9% for combination; P = 0.015) but not for BxPC-3. FACS analysis showed that the expressed EGFR level decreased by about 42% on MiaPaCa 2 whereas no loss was seen on BxPC-3. Expression of BAX was upregulated on MiaPaCa-2. Poly (ADP-ribose) polymerase cleavage indicated the killing was mediated by apoptosis. Immunoblots showed that Grb2 was co-immunoprecipitated with EGFR after EGF stimulation. Incubation with Erbitux blocked Grb2 binding in MiaPaCa-2 but not BxPC 3. FGF transactivated EGFR down stream Ras-MAPK in the presence or absence of Erbitux. Internalization of EGFR induced by Erbitux did not differ between MiaPaCa-2 and BxPC-3. CONCLUSIONS: 1) Association of Grb2 to EGFR in BxPC-3 induced by EGF in the presence of Erbitux indicates an alternate pathway of Ras-MAPK activation, which may be related with the tumor resistance to treatment; 2) transactivation of EGFR downstream Ras-MAPK pathway by FGF contributes the resistance to treatment; and 3) downregulation of EGFR may increase the response to therapy.

Adaptor Proteins, Signal Transducing↗
PubMed2003

An adenovirus encoding proapoptotic Bax synergistically radiosensitizes malignant glioma.

PURPOSE: We explore the utility of the adenovirus-mediated delivery of proapoptotic Bax for enhancing the cytotoxicity of radiotherapy (RT) in RT-refractory glioma cells. MATERIALS AND METHODS: Cell lines D54 MG and U87 MG (p53 wild-type), and U251 MG and U373 MG (p53 mutant), and patient-derived astrocytes were evaluated. Cells were irradiated and infected with an inducible adenovirus encoding Bax. Cell proliferation, colony formation assay, quantification of early apoptotic alteration in the plasma membrane by fluorescence-activated cell sorter using annexin V, and nuclear staining with H33258 were used to evaluate apoptosis. The capacity of the combined treatment to induce regression of subcutaneous D54 MG tumors was tested in nude mice. A dose of 5 Gy was administered every other day, four times, for a total dose of 20 Gy. One day after each irradiation, tumors were injected with 1 x 10(9) plaque-forming units (PFU). RESULTS: Apoptotic death was enhanced by the combination of Ad/Bax and RT. In D54 MG, levels of apoptosis after RT alone, Ad/Bax alone, or the combination were, respectively, 12.3%, 32.1%, and 78.5%. In contrast, treatment of astrocytes did not significantly induce apoptosis. A colony-formation assay showed a 2-log inhibition with respect to controls after combined treatment, irrespective of the endogenous levels of p53. The other apoptosis assays also showed the defining characteristics of apoptosis in the combination group. Remarkably, combined treatment induced regression of tumors in mice. CONCLUSIONS: Ad/Bax synergistically radiosensitizes glioma, with a seemingly favorable therapeutic index.

Adenoviridae↗
PubMed2003

Three-dimensional dose model for the comparison of 177Lu-HuCC49DeltaCH2 and 177Lu-HuCC49 radioimunotherapy in mice bearing intraperitoneal xenografts.

Uptake and dose distributions in peritoneal LS174T colon tumor xenografts were compared for a humanized construct of the CC49 (HuCC49) high-affinity anti-TAG-72 monoclonal antibody and a construct with the CH2 region deleted (HuCC49DeltaCH2), both labeled with (177)Lu using a PA-DOTA bifunctional chelating agent and injected in the peritoneum. Tumors were resected and serially sectioned at 1 h, 4 h, 24 h, and 48 h postinjection. Between 5 and 24 (average 16) sections were retained per tumor for autoradiography. The typical section interval was 340 microm and thickness was 16 microm. Tumor sections were air dried and placed on film and/or phosphor screen. Section images were digitized at 100 microm resolution electronically (phosphor screen) or by laser densitometer (film). Section images were used to generate tumor surface descriptions and activity distributions by reconstructing the activity densities in three dimensions. Three-dimensional dose-rate calculations, performed using a point kernel for (177)Lu, were used to prepare radial histograms describing the variation in dose rate as a function of distance from the tumor center to surface. At early times postinjection, the (177)Lu-HuCC49DeltaCH2 antibody displayed higher dose rates near the tumor surface compared to the (177)Lu-HuCC49 antibody. At 24 h postinjection, dose rate distributions appeared similar for both antibodies and more uniform than at earlier times. The (177)Lu-HuCC49DeltaCH2 antibody indicated improved uniformity at 48 h postinjection. Cell survival calculations based on the three-dimensional dose rate distributions favored (177)Lu-HuCC49DeltaCH2 for equal injection activities. However the most significant effect was the greater injected dose tolerated for the (177)Lu-HuCC49DeltaCH2 antibody based on equivalent estimated bone marrow dose.

Aniline Compounds↗
PubMed2002

Treatment of pancreatic cancer xenografts with Erbitux (IMC-C225) anti-EGFR antibody, gemcitabine, and radiation.

PURPOSE: To investigate treatment of human pancreatic cancer cell lines and xenografts with combinations of Erbitux (IMC-C225) anti-epidermal growth factor receptor (EGFR) antibody, gemcitabine, and radiation. METHODS AND MATERIALS: BxPC-3 and MiaPaCa-2 human pancreatic carcinoma cells were treated in vitro for 24 h with IMC-C225 (5 microg/mL), then exposed to epidermal growth factor (EGF) (10 mM) for 5 min. Immunoblots were screened for EGFR expression and the ability of IMC-C225 to block EGF-induced tyrosine phosphorylation of EGFR. Cells were treated with IMC-C225 (5 microg/mL) on Day 0, the IC(50) dose of gemcitabine on Day 1 for 24 h, followed by 3 Gy 60Co irradiation on Day 2, or the combination of each agent. For cell proliferation, cells were counted on Day 4, and for apoptosis, cells were stained with annexin V-FITC and propidium iodide, then analyzed by FACS. Cells were treated with the same single or multiple treatments and analyzed in a clonogenic cell survival assay. The effect of IMC-C225, gemcitabine, and radiation on the growth of BxPC-3 and MiaPaCa-2 tumor xenografts was determined. Athymic nude mice bearing established s.c. tumor xenografts of 6-8 mm diameter received 6 weeks of treatment with IMC-C225 (1 mg every 3 days x 6) alone or in combination with gemcitabine (120 mg/kg i.v. every 6 days x 6), and 6 weekly fractions of 3 Gy radiation on the days after gemcitabine administration. Tumor growth was measured with Vernier calipers. RESULTS: BxPC-3 and MiaPaCa-2 cell lines expressed low levels of EGFR. IMC-C225 inhibited EGF-induced tyrosine phosphorylation of the EGF receptor on both cell lines. Treatment of cells with a combination of IMC-C225 + gemcitabine + radiation produced the highest induction of apoptosis and inhibition of proliferation in vitro. Combination treatment with IMC-C225, gemcitabine, and radiation produced 100% complete regression of MiaPaCa-2 tumors for more than 250 days, and the greatest growth inhibition of BxPC-3 tumors compared to any single or dual treatments. CONCLUSIONS: The IMC-C225 therapy in combination with gemcitabine chemotherapy and radiation therapy demonstrated statistically significantly greater efficacy over the single and double combination therapies. This form of multimodality treatment shows potential clinical application in the treatment of pancreatic cancer in humans.

Animals↗
PubMed2002

Model prediction of treatment planning for dose-fractionated radioimmunotherapy.

BACKGROUND: Clinical trials of radioimmunotherapy (RIT) often use dose fractionation to reduce marrow toxicity. The dosing scheme can be optimized if marrow and tumor cell kinetics following radiation exposure are known. METHODS: A mathematic model of tumor clonogenic cell kinetics was combined with a previously reported marrow cell kinetics model that included marrow stromal cells, progenitor cells, megakaryocytes, and platelets. Reported values for murine tumor and marrow cellular turnover rates and radiosensitivity were used in the model calculation. RESULTS: Given a tolerated level of thrombocytopenia, there is a fractionation scheme in which total radioactive dose administration can be maximized. Isoeffect doses that had different numbers of fractions and total radioactivity, but induced identical platelet nadirs of 20%, were determined. Assuming identical tumor uptake for all dose fractions, six tumor types were examined: early-responding tumors, late-responding tumors, and tumors that lacked a late-responding effect, with either constant or accelerated doubling time. For most tumor types, better tumor control (tumor growth delay and nadir of survival fraction) was predicted for a dosing scheme in which total radioactive dose was maximized. For late-responding tumors with accelerated doubling time, tumor growth delay increased, but the nadir of survival fraction became shallower as the number of fractions increased. CONCLUSIONS: A mathematic model has been developed that allows prediction of the nadir and duration of thrombocytopenia as well as tumor clonogenic cell response to various RIT doses and fractionation schemes. Given a maximum tolerated level of thrombocytopenia, the model can be used to determine a dosing scheme for optimal tumor response.

Abnormalities, Radiation-Induced↗
PubMed2002

Targeted radiotherapy with [(90)Y]-SMT 487 in mice bearing human nonsmall cell lung tumor xenografts induced to express human somatostatin receptor subtype 2 with an adenoviral vector.

BACKGROUND: Novel approaches to increasing the therapeutic efficacy of targeted radiotherapy of cancer are required. One strategy to achieve this goal is to induce high-level expression of a receptor on the surface of tumor cells that can be targeted with a radiolabeled peptide. The objectives of this study were to 1) induce somatostatin receptor (SSTr2) expression in tumor cells using an adenovirus encoding the SSTr2 gene (AdSSTr2), 2) demonstrate tumor localization of [(111)In]-DTPA-D-Phe(1)-octreotide in AdSSTr2-injected tumors, and 3) show therapeutic efficacy with [(90)Y]-DOTA-D-Phe(1)-Tyr(3)-octreotide ([(90)Y]-SMT 487). METHODS: SSTr2 expression was validated in vitro by the binding and subsequent internalization of [(111)In]-DTPA-D-Phe(1)-octreotide (21.3% per mg of total protein) in A-427 cells infected with AdSSTr2. In vivo imaging confirmed 5- to 10-fold greater uptake 5.5 hours after intravenous administration of [(111)In]-DTPA-D-Phe(1)-octreotide in AdSSTr2-injected tumors relative to control tumors. For therapy studies, mice bearing established subcutaneous A-427 tumors were given two intratumoral injections of AdSSTr2 1 week apart, followed by an intravenous injection of 400 microCi or 500 microCi [(90)Y]-SMT 487 at 2 and 4 days after each adenoviral administration. Control animals either were not treated or were administered 500 microCi [(90)Y]-SMT 487 with no AdSSTr2 injection. RESULTS: These studies showed that untreated animals and animals treated with no virus and 500 microCi [(90)Y]-SMT 487 had median tumor quadrupling times of 16 and 25 days, respectively. Mice administered AdSSTr2 and either 400 microCi or 500 microCi of [(90)Y]-SMT 487 demonstrated significantly longer median tumor quadrupling times of 40 and 44 days, respectively (P < 0.02). CONCLUSIONS: These studies are the first to demonstrate in vivo therapeutic efficacy using a radiolabeled peptide targeted to a receptor expressed on the surface of tumor cells following gene transfer. Future studies will focus on the optimization of this approach.

Adenoviridae↗
PubMed2002

Rationales, evidence, and design considerations for fractionated radioimmunotherapy.

Although fractionation can be used in a discrete radiobiologic sense, herein it is generally used in the broader context of administration of multiple, rather than single, doses of radionuclide for radioimmunotherapy (RIT) or other targeted radionuclide therapies. Fractionation is a strategy for overcoming heterogeneity of monoclonal antibody (MAb) distribution in the tumor and the consequent nonuniformity of tumor radiation doses. Additional advantages of fractionated RIT are the ability to 1) provide patient-specific radionuclide and radiation dosing, 2) control toxicity by titration of the individual patient, 3) reduce toxicity, 4) increase the maximum tolerated dose (MTD) for many patients, 5) increase tumor radiation dose and efficacy, and 6) prolong tumor response by permitting treatment over time. However, fractionated RIT has logistic and economic implications. Preclinical and clinical data substantiate the advantages of fractionated RIT, although the radiobiology for conventional external beam radiotherapy does not provide a straightforward rationale for RIT unless fractionation leads to more uniform distribution of radiation dose throughout the tumor. Preclinical data have shown that toxicity and mortality can be reduced while efficacy is increased, thereby providing inferential evidence of greater uniformity of radiation dose. Direct evidence of superior dosimetry and tumor activity distribution has also been found. Clinical data have shown that toxicity can be better controlled and reduced and the MTD extended for many patients. It is clear that fractionated RIT can only fulfill its potential if the effects of critical issues, such as the number and amount of radionuclide doses, the radionuclide physical and effective half-life, and the dose interval, are better characterized.

Animals↗
PubMed2002

Quantitation of cytosine deaminase mRNA by real-time reverse transcription polymerase chain reaction: a sensitive method for assessing 5-fluorocytosine toxicity in vitro.

Cytosine deaminase/5-fluorocytosine (CD/5-FC) is a promising strategy for local cancer gene therapy. We hypothesized that CD expression within tumor cells would be directly related to efficacy and that quantitation of markers of CD expression such as mRNA, protein, and enzyme activity would therefore facilitate prediction of 5-FC toxicity. These three markers were thus quantitated by real-time quantitative reverse transcription polymerase chain reaction (Q-RT-PCR), semiquantitative immunocytochemistry (ICC), and 5-[(3)H]FC enzyme assay, respectively. Results with human colon (LS174T) cancer cells infected with a replication-incompetent adenovirus encoding CD (AdCMVCD) demonstrated a significant correlation between CD mRNA and enzyme activity up to 24 h postinfection. A direct correlation was found between CD dose (AdCMVCD PFU/cell) and CD mRNA and protein expression (P < 0.002) in both LS174T and BxPC-3 pancreatic cancer cells, but the relationship with enzyme activity was less strong in LS174T cells (P = 0.09). A remarkable concordance existed among Q-RT-PCR, ICC and enzyme assays with both cell lines. Importantly, CD dose and mRNA and protein expression inversely correlated with 5-FC IC(50) (P < 0.02). Quantitation of CD markers also facilitated identification of factors governing differential susceptibility to CD/5-FC. These results suggest that Q-RT-PCR will be useful for monitoring transgene expression in future studies using improved CD-based expression vectors and may also be useful in predicting the response to CD/5-FC therapy, which is likely to be heterogeneous in the patient population.

Antimetabolites↗
PubMed2002

Intratumoral 5-fluorouracil produced by cytosine deaminase/5-fluorocytosine gene therapy is effective for experimental human glioblastomas.

5-Fluorouracil (5-FU) is a potent antimetabolite used for chemotherapy of gastrointestinal (GI), breast, and head and neck malignancies. Although clinical trials have been conducted, the poor therapeutic index of 5-FU has precluded its clinical use for a number of other tumor types. It is unclear whether this lack of utility is due to problems with drug delivery or inherent insensitivity. Adenovirus (Ad) vector-mediated cytosine deaminase (CD)/5-fluorocytosine (5-FC) gene therapy has the potential to overcome pharmacokinetic issues associated with systemic 5-FU and is particularly well suited to use with tumors in which local control is paramount, such as recurrent, localized prostate cancer and malignant gliomas. In this study, the in vitro response by a panel of human tumor cell lines derived from both GI (colon, pancreas) and non-GI (prostate, glioma) tumors to 5-FU and to AdCMVCD (an Ad encoding Escherichia coli CD)/5-FC was examined. Whereas the sensitivity (IC(50)) of individual cell lines to these agents varied, no significant difference in median IC(50) for either 5-FU or AdCMVCD/5-FC was evident for the four tumor types tested (P > 0.1). The relevant contributions of Ad gene transfer efficiency and inherent 5-FU sensitivity in determining response to AdCMVCD/5-FC were then assessed. Multiple linear regression analysis revealed that whereas both factors significantly contribute to the response, inherent 5-FU sensitivity was substantially more important (beta= 0.78 versus 0.48; P < 0.001). Finally, the therapeutic efficacy of a single intratumoral injection of AdCMVCD followed by systemic 5-FC was assessed in three intracranial C.B17 severe combined immunodeficient mouse models of human glioma. AdCMVCD/5-FC efficacy was specific, virus dose-dependent, and closely paralleled in vitro 5-FU and CD/5-FC sensitivity in two of three models tested. These results reveal that glioma cells are as sensitive as GI tumor cells to the antineoplastic effects of 5-FU, identify inherent 5-FU sensitivity as an important factor in determining CD/5-FC efficacy, and confirm previous findings in rat models that demonstrate the potential clinical utility of AdCMVCD/5-FC gene therapy for gliomas.

Adenoviridae↗
PubMed2002

Adenovirus-mediated transfer of BAX driven by the vascular endothelial growth factor promoter induces apoptosis in lung cancer cells.

Apoptosis induction is a promising approach for cancer gene therapy. Bax is a death-promoting member of the Bcl2 family of genes that are intimately involved in apoptosis. Overexpression of BAX protein can accelerate cell death by homodimers that promote apoptosis in a variety of cancer cell lines. The cytotoxic effect of BAX was evaluated in vitro by a recombinant adenovirus system expressing the human BAX gene under control of human vascular endothelial growth factor (VEGF) promoter element (AdVEGFBAX). Overexpression of BAX in human lung carcinoma cells resulted in apoptosis induction, caspase activation, and cell growth suppression, none of which were observed in BEAS-2B normal human bronchial epithelial cells that do not overexpress VEGF under normoxic conditions. To examine the hypoxia responsiveness of the VEGF promoter, lung cancer cells were transiently exposed to hypoxia; this treatment increased enhanced green fluorescent protein (EGFP) expression after AdVEGFEGFP infection in both normal and cancer cell lines, and enhanced apoptosis and decreased the number of surviving cancer cells compared with the Ad/BAX plus Ad/Cre binary adenoviral system. These results suggest a possible therapeutic application of cancer-specific expression of the pro-apoptotic Bax gene driven by the VEGF promoter.

Adenoviridae↗
PubMed2002

Gamma camera dual imaging with a somatostatin receptor and thymidine kinase after gene transfer with a bicistronic adenovirus in mice.

PURPOSE: To compare two systems for assessing gene transfer to cancer cells and xenograft tumors with noninvasive gamma camera imaging. MATERIALS AND METHODS: A replication-incompetent adenovirus encoding the human type 2 somatostatin receptor (hSSTr2) and the herpes simplex virus thymidine kinase (TK) enzyme (Ad-hSSTr2-TK) was constructed. A-427 human lung cancer cells were infected in vitro and mixed with uninfected cells at different ratios. A-427 tumors in nude mice (n = 23) were injected with 1 x 10(6) to 5 x 10(8) plaque-forming units (pfu) of Ad-hSSTr2-TK. The expressed hSSTr2 and TK proteins were imaged owing to internally bound, or trapped, technetium 99m ((99m)Tc)-labeled hSSTr2-binding peptide (P2045) and radioiodinated 2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl-5-iodouracil (FIAU), respectively. Iodine 125 ((125)I)-labeled FIAU was used in vitro and iodine 131 ((131)I)-labeled FIAU, in vivo. The (99m)Tc-labeled P2045 and (125)I- or (131)I-labeled FIAU were imaged simultaneously with different window settings with an Anger gamma camera. Treatment effects were tested with analysis of variance. RESULTS: Infected cells in culture trapped (125)I-labeled FIAU and (99m)Tc-labeled P2045; uptake correlated with the percentage of Ad-hSSTr2-TK-positive cells. For 100% of infected cells, 24% +/- 0.4 (mean +/- SD) of the added (99m)Tc-labeled P2045 was trapped, which is significantly lower (P <.05) than the 40% +/- 2 of (125)I-labeled FIAU that was trapped. For the highest Ad-hSSTr2-TK tumor dose (5 x 10(8) pfu), the uptake of (99m)Tc-labeled P2045 was 11.1% +/- 2.9 of injected dose per gram of tumor (thereafter, dose per gram), significantly higher (P <.05) than the uptake of (131)I-labeled FIAU at 1.6% +/- 0.4 dose per gram. (99m)Tc-labeled P2045 imaging consistently depicted hSSTr2 gene transfer in tumors at all adenovirus doses. Tumor uptake of (99m)Tc-labeled P2045 positively correlated with Ad-hSSTr2-TK dose; (131)I-labeled FIAU tumor uptake did not correlate with vector dose. CONCLUSION: The hSSTr2 and TK proteins were simultaneously imaged following dual gene transfer with an adenovirus vector.

Adenoviridae↗
PubMed2002

Induction of thymidine phosphorylase in both irradiated and shielded, contralateral human U87MG glioma xenografts: implications for a dual modality treatment using capecitabine and irradiation.

In the United States, tumors of the central nervous system remain the third leading cancer-related cause of death in young adults with a median survival time of < 1 year. A recent case study suggested that Capecitabine (a novel, fluoropyrimidine prodrug) may be effective in the treatment of brain metastases. Pharmacogenomic studies have correlated the antitumor response to Capecitabine with the expression of the drug metabolizing enzymes thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD). In the current study, we examined TP and DPD expression in normal human brain tissues and in glioblastoma multiforme, the most common and malignant type of brain tumor. Because previous reports suggest a tumor necrosis factor (TNF)-alpha-mediated increase in TP expression after irradiation (a current standard of care for glioblastoma multiforme), we also examined the effect of irradiation on the expression of TP, DPD, and TNF-alpha in both irradiated and lead-shielded contralateral U87MG glioma xenografts within the same animal. Expression levels were determined using real-time quantitative PCR as described previously. Results demonstrate an approximately 70-fold increase in TP mRNA levels 4 days after irradiation, relative to initial control levels. Interestingly, TP mRNA in the lead-shielded tumors (contralateral to irradiated tumors) increased approximately 60-fold by day 10 relative to initial control levels. Elevated TP levels were sustained for 20 days in irradiated xenografts but began to decrease after 15 days in the shielded/contralateral tumors, returning to baseline by 20 days. TP mRNA levels in normal mouse liver were unaltered, suggesting a tumor-associated effect. TNF-alpha mRNA levels did not increase after irradiation; therefore, mRNA expression of 11 additional cytokines [interleukin (IL)-1 alpha, IL-1 beta, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p35, IL-12p40, IL-15, and IFN-gamma] in both the irradiated and shielded xenografts was quantitated. Results demonstrated increased levels of IFN-gamma, IL-10, and IL-1 alpha by 6.3-, 3.7-, and 1.6-fold, respectively, in irradiated tumors only. DPD mRNA levels did not change after irradiation. The tumor-associated induction of TP in irradiated and lead-shielded tumors within the same animal may have significant implications for the combined modality treatment of cancer patients with Capecitabine in conjunction with radiotherapy and may apply to the treatment of distant tumors and or metastatic disease.

Animals↗
PubMedDate not supplied

De novo synthesis of a new diethylenetriaminepentaacetic acid (DTPA) bifunctional chelating agent.

Diethylene triamine pentaacetic acid (DTPA) has been in extensive use as a metal chelator in the development of radiopharmaceuticals and contrast agents. The former application uses DTPA mostly as a bifunctional chelating agent (BCA) conjugated to tumor-targeting vehicles (TTVs) such as monoclonal antibodies (MAbs) and receptor-directed peptides. A new bifunctional DTPA derivative was synthesized by a fully organic scheme. This compound, N(4),N(alpha),N(alpha),N(epsilon),N(epsilon)-[pentakis(carboxymethyl)]-N(4)-(carboxymethyl)-2,6-diamino-4-azahexanoic hydrazide (20) was prepared by a convergent synthesis strategy using N(alpha)-benzyloxycarbonyl-2,3-diaminopropionic acid as the starting compound. This commercially available material was used to build a functionalized triamine which served as the molecular core template for assembling the target molecule. To evaluate the conjugation and radiolabeling capabilities of this new molecule, it was covalently attached to the anti-TAG-72 MAb, Delta CH2HuCC49, and the conjugate was radiolabeled in near-quantitative yields with yttrium-90 ((90)Y) and lutetium-177 ((177)Lu). Biodistribution of the (177)Lu-labeled DTPA-Delta CH2HuCC49 in tumor-bearing nude mice demonstrated preservation of the immunoreactivity of the MAb as indicated by high tumor uptake. In addition to the introduction of a new bifunctional DTPA, this work reports on a novel synthetic approach for preparation of this useful metal chelator and introduces a new conjugation protocol.

Animals↗
PubMedDate not supplied

Synthesis of the first diethylenetriaminepentahydroxamic acid (DTPH) bifunctional chelating agent.

Synthesis of a new pentahydroxamic acid bifunctional chelating agent (BCA), constructed on the aminoazaalkyl core of diethylenetriaminepentaacetic acid (DTPA), is reported. Rational modifications in the structure of DTPA, which could result in an enhancement of its chelation properties, add to the collection of diagnostic and therapeutic metals bound by this chelator, and might implement significant improvements in the in vivo behavior of this compound, are described. Further improvements in the stability of the ligand-metal complexes of DTPA may improve both diagnostic and therapeutic outcomes such as tumor-to-normal tissue ratios and target-delivered radioactivity. A combination of hydroxamate functions with the azaalkyl backbone of DTPA might be a suitable approach to generate such higher stabilities. This rationale may be justified by the well-known affinity of hydroxamates against different transition metals and favorable properties of DTPA as a versatile chelator. Thus, the N(4),N(alpha),N(alpha),N(epsilon),N(epsilon)-pentakis[[((N-hydroxy-N-methyl]carbonyl)methyl]-2, 6-diamino-4-azahexanoic hydrazide (5, DTPH) was designed and synthesized through a convergent synthesis and in 40.7% overall yield. Conjugation of this compound to the monoclonal antibody (MAb) Delta Ch2HuCC49, used as a model protein, was carried out to evaluate the efficiency of this molecule as a BCA. Radiolabeling of the DTPH-Delta CH2HuCC49 conjugate with lutetium-177 ((177)Lu) and biodistribution of the labeled conjugate in athymic nude mice, bearing LS174T human colon carcinoma xenografts, are reported.

Animals↗
PubMedDate not supplied

Synthesis and biological evaluation of paclitaxel-C225 conjugate as a model for targeted drug delivery.

Tumor-targeted drug delivery is an attractive strategy in cancer treatment. We have previously reported a paclitaxel model conjugate using a bombesin receptor-recognizing peptide in which the drug cytotoxicity against H1299 human nonsmall cell lung cancer was enhanced compared to unconjugated taxol. In an effort to expand the development of tumor-recognizing taxanes, paclitaxel (PTX, taxol) was conjugated to the anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody (MAb) Erbitux (C225) to serve as a model MAb-mediated drug delivery compound. Thus, paclitaxel was derivatized at its 2'-hydroxy function by introduction of a succinate linker, and the carboxyl group of the latter was covalently attached to C225 through amide bond formation. The final product conjugate (PTXC225) was analyzed mass spectrometrically for assessment of the drug-to-antibody ratios. Cytotoxicity screening of the drug-antibody conjugate against A431, UM-SCC-1, and UM-SCC-6 cells indicated an enhancement in cytocidal effect of paclitaxel as compared to those of the free drug, the intact antibody, and a physical mixture of the two (the controls). In A431 cells, the conjugate showed 25.2% +/- 2.2% of apoptosis induction as compared to little or no apoptosis caused by the controls. Biodistribution analysis of the PTXC225 in tumor-implanted nude mice and a tyrosine-kinase assay showed that conjugation of the drug did not interfere with the immunoreactivity of the antibody. The 24-h tumor uptake of C225 and PTXC225 were 11.7% +/- 6.0% and 7.1% +/- 3.6% of the injected dose per gram of tissue (%ID/g), respectively, which were not significantly different. Also, in A431-implanted nude mice, the conjugate and C225 showed tumor growth inhibition effects of 57.2% and 41.2%, respectively, against a saline-treated control, which were not significantly different from each other. This lack of difference in the in vivo antitumor activity of the MAb-delivered drug and free PTX may be due to either a relatively low dose of the antibody-delivered drug (346 microg/kg), or an untimely release of it, or both. The tumor growth inhibition pattern of the conjugate, however, was identical to that of C225, indicating that the attachment of PTX did not affect the antigen-binding and growth inhibitory features of the MAb. These preliminary results demonstrate the potential of tumor-targeted delivery of taxol as a promising strategy in cancer treatment and warrant further work to develop more suitable drug-MAb linkers as well as improved dosage and treatment protocols.

Algorithms↗
PubMedDate not supplied

Site-specifically traced drug release and biodistribution of a paclitaxel-antibody conjugate toward improvement of the linker structure.

Tumor-directed drug delivery is a promising strategy in cancer treatment, and in this field, monoclonal antibodies constitute an important class of targeting vehicles. A critical issue in the design of targeting conjugates is the timing of the release of the cytotoxic payload, with the ideal situation being the release at the maximum tumor uptake of the targeting molecule. A site-specific radiolabeling technique was used to elucidate the biodistribution and in vivo drug release pattern of an antibody conjugate of paclitaxel (PTX, 1, Figure 1) in which the drug and the antibody moieties were connected by a succinate (SX) linker. In this new method, a metabolite of PTX, 3'-(4-hydroxyphenyl)paclitaxel (3'-OH-PTX, 2, Figure 1) was used as a tyrosine mimic for the synthesis of the drug site-labeled conjugate (DSL, [(125)I]-3'-OH-PTXSXC225). This was achieved by iodogen (125)I-labeling of 3'-OH-PTXSX and subsequent conjugation to C225. The antibody site-labeled conjugate (ASL, PTXSX-[(125)I]-C225) was prepared by direct radioiodination of PTXSXC225. Biodistribution of these compounds was studied in Balb/c nude mice bearing DU-145 human prostate carcinoma xenografts. While the 4 and 24 h tumor uptake (in percent injected dose per gram of tissue, %ID/g) for [(125)I]-3'-OH-PTXSXC225 were 3.3 +/- 1.5 and 1.7 +/- 0.6%ID/g, the PTXSX-[(125)I]-C225 showed tumor uptake values of 3.8 +/- 4.2 and 14.8 +/- 4.2%ID/g at these time points. This difference in the tumor uptake over time indicates an early cleavage of the drug with respect to the antibody tumor localization. This was further confirmed by an in vitro drug release kinetics study leading to a half-life of about 2 h for PTXSXC225 under physiological conditions. To increase the stability of the PTX-MAb bond, a new conjugate (PTXGLC225) with glutaric acid (GL) as the linker was synthesized. Under the same conditions, the PTXGLC225 showed a 16-fold increase in the half-life (t(1/2)) of the drug release. The effect of the increased t(1/2) of this compound on the antitumor activity of the conjugate was tested in a DU-145 human prostate tumor-implanted mouse model. In comparison to a previous similar experiment with PTXSXC225, better antitumor activity was observed for the PTXGLC225 conjugate as compared to controls. These results demonstrated the first time use of radioiodinated 3'-OH-PTX for in vivo tracing of a paclitaxel conjugate and application of the resulting information to the design of a therapeutically more useful PTX-MAb linker.

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