Zbigniew P. Kortylewicz

Effect of Platelet-Derived Growth Factor Receptor-β Inhibition with STI571 on Radioimmunotherapy

Whereas radioimmunotherapy of hematologic malignancies has evolved into a viable treatment option, the responses of solid tumors to radioimmunotherapy are discouraging. The likely cause of this problem is the interstitial hypertension inherent to all solid tumors. Remarkable improvements in tumor responses to radioimmunotherapy were discovered after the inclusion of STI571 in the therapy regimen. A combination of the tumor stroma-reactive STI571, a potent platelet-derived growth factor receptor-beta (PDGFr-beta) antagonist, and the tumor-seeking radiolabeled antibody B72.3 yielded long-lasting growth arrest of the human colorectal adenocarcinoma LS174T grown as s.c. xenografts in athymic mice. The interaction of STI571 with the stromal PDGFr-beta reduced tumor interstitial fluid pressure (P(IF)) by >50% and in so doing improved the uptake of B72.3. The attenuation of P(IF) also had a positive effect on the homogeneity of antibody distribution. These effects were dose-dependent and under optimized dosing conditions allowed for a 2.45 times increase in the tumor uptake of B72.3 as determined in the biodistribution studies. Single-photon emission computed tomography imaging studies substantiated these results and indicated that the homogeneity of the radioisotope distribution was also much improved when compared with the control mice. The increased uptake of radioimmunotherapy into the tumor resulted in >400% increase in the tumor absorbed radiation doses in STI571 + radioimmunotherapy-treated mice compared with PBS + radioimmunotherapy-treated mice. The improved antibody uptake in response to the attenuation of tumor P(IF) was identified as the primary reason for the growth arrest of the STI571 + radioimmunotherapy-treated tumors. Two related causes were also identified: (a) the improved homogeneity of monoclonal antibody distribution in tumor and (b) the increased tumor radiosensitivity resulting from the improved tumor oxygenation.

Radiolabeled 5-Iodo-3′-O-(17β-succinyl-5α-androstan-3-one)-2′-deoxyuridine and Its 5′-Monophosphate for Imaging and Therapy of Androgen Receptor-Positive Cancers: Synthesis and Biological Evaluation

High levels of androgen receptor (AR) are often indicative of recurrent, advanced, or metastatic cancers. These conditions are also characterized by a high proliferative fraction. 5-Radioiodo-3-O-(17beta-succinyl-5alpha-androstan-3-one)-2-deoxyuridine 8 and 5-radioiodo-3-O-(17beta-succinyl-5alpha-androstan-3-one)-2-deoxyuridin-5-yl monophosphate 13 target AR. They are also degraded intracellularly to 5-radioiodo-2-deoxyuridine 1 and its monophosphate 20, respectively, which can participate in the DNA synthesis. Both drugs were prepared at the no-carrier-added level. Precursors and methods are readily adaptable to radiolabeling with various radiohalides suitable for SPECT and PET imaging, as well as endoradiotherapy. In vitro and in vivo studies confirm the AR-dependent interactions. Both drugs bind to sex hormone binding globulin. This binding significantly improves their stability in serum. Biodistribution and imaging studies show preferential uptake and retention of 8 and 13 in ip xenografts of human ovarian adenocarcinoma cells NIH:OVCAR-3, which overexpress AR. When these drugs are administered at therapeutic dose levels, a significant tumor growth arrest is observed.

Radiolabeled cyclosaligenyl monophosphates of 5-iodo-2'-deoxyuridine, 5-iodo-3'-fluoro-2',3'-dideoxyuridine, and 3'-fluorothymidine for molecular radiotherapy of cancer: synthesis and biological evaluation.

Targeted molecular radiotherapy opens unprecedented opportunities to eradicate cancer cells with minimal irradiation of normal tissues. Described in this study are radioactive cyclosaligenyl monophosphates designed to deliver lethal doses of radiation to cancer cells. These compounds can be radiolabeled with SPECT- and PET-compatible radionuclides as well as radionuclides suitable for Auger electron therapies. This characteristic provides an avenue for the personalized and comprehensive treatment strategy that comprises diagnostic imaging to identify sites of disease, followed by the targeted molecular radiotherapy based on the imaging results. The developed radiosynthetic methods produce no-carrier-added products with high radiochemical yield and purity. The interaction of these compounds with their target, butyrylcholinesterase, depends on the stereochemistry around the P atom. IC(50) values are in the nanomolar range. In vitro studies indicate that radiation doses delivered to the cell nucleus are sufficient to kill cells of several difficult to treat malignancies including glioblastoma and ovarian and colorectal cancers.

Colon-specific prodrugs of 5-radioiodo-2'-deoxyuridine

Two glycoside-based prodrugs, 125IUdR-5-beta-D-glucopyranoside and 125IUdR-5-beta-D-galactopyranoside, were synthesized. This selection was dictated by the abundance of appropriate enzymes in the GI tract of mice and similar levels of beta-D-glycosidases in human and rodent large intestine. Studies to establish the ability of colonic microflora to release 125IUdR were conducted in vitro and in Swiss Webster mice. Both prodrugs released 125IUdR in the presence of the corresponding enzymes or the GI content homogenates in vitro, and in vivo. Luminal enzymes in the proximal and distal small intestine in mice degraded less than 10% of each prodrug whereas enzymes from the colonic/caecal lumen of mice released nearly 100% of 125IUdR. 125IUdR freed by bacterial glycosidases was stable in the GI content. No significant amounts of other metabolites or deiodination products were observed. Total radioactivity recovered as by-products was less than 10%. The efflux of prodrugs from the GI tract after oral administration in mice was slow and limited. Unlike 125IUdR, prodrugs were not dehalogenated in vivo as indicated by biodistribution and imaging studies.

Co‐targeting androgen receptor and DNA for imaging and molecular radiotherapy of prostate cancer: In vitro studies

The androgen receptor (AR) axis, the key growth and survival pathway in prostate cancer, remains a prime target for drug development. 5‐Radioiodo‐3′‐O‐(17β‐succinyl‐5α‐androstan‐3‐one)‐2′‐deoxyuridin‐5′‐yl phosphate (RISAD‐P) is the AR‐seeking reagent developed for noninvasive assessment of AR and proliferative status, and for molecular radiotherapy of prostate cancer with Auger electron‐emitting radionuclides.

Butyrylcholinesterase as a Blood Biomarker in Neuroblastoma.

Blood-based biomarkers are important in the detection of the disease and in the assessment of responses to therapy. In this study, butyrylcholinesterase was evaluated as a potential biomarker in newly diagnosed neuroblastoma (NB) patients at diagnosis and longitudinally during treatment. Plasma butyrylcholinesterase activities in age-matched and sex-matched children were used as controls. Pretreatment butyrylcholinesterase levels in NB subjects are on an average 2 times lower than butyrylcholinesterase levels in healthy subjects. Significantly, butyrylcholinesterase activities are ∼40% lower in MYCN-amplified as compared with nonamplified disease. As the course of chemotherapy progresses, butyrylcholinesterase activities recover and normalize to control values. The evident response to treatment indicates that plasma butyrylcholinesterase is a good biomarker of tumor response to therapy. Depressed butyrylcholinesterase levels in NB subjects are not caused by hepatic deficits suggesting a specific role for butyrylcholinesterase in NB. Further examination of the mechanism of altered butyrylcholinesterase production require an animal model that best approximates human condition. Studies in mice show that murine NB allografts significantly reduce butyrylcholinesterase activity in plasma. This finding correlates with changes observed in NB patients. In contrast, human NB xenografts produce the opposite effect, that is, butyrylcholinesterase plasma levels rise as the xenograft size increases. In the absence of any liver damage, dissimilarities between butyrylcholinesterase production in murine and human NB models suggest species-specific signaling pathways. This disparity also suggests that human NB xenograft mouse models do not approximate the human disease.

Activation of PDGFr-β Signaling Pathway after Imatinib and Radioimmunotherapy Treatment in Experimental Pancreatic Cancer.

Pancreatic cancer does not respond to a single-agent imatinib therapy. Consequently, multimodality treatments are contemplated. Published data indicate that in colorectal cancer, imatinib and radioimmunotherapy synergize to delay tumor growth. In pancreatic cancer, the tumor response is additive. This disparity of outcomes merited further studies because interactions between these modalities depend on the imatinib-induced reduction of the tumor interstitial fluid pressure. The examination of human and murine PDGFr-β/PDGF-B pathways in SW1990 pancreatic cancer xenografts revealed that the human branch is practically dormant in untreated tumors but the insult on the stromal component produces massive responses of human cancer cells. Inhibition of the stromal PDGFr-β with imatinib activates human PDGFr-β/PDGF-B signaling loop, silent in untreated xenografts, via an apparent paracrine rescue pathway. Responses are treatment-and time-dependent. Soon after treatment, levels of human PDGFr-β, compared to untreated tumors, are 3.4×, 12.4×, and 5.7× higher in imatinib-, radioimmunotherapy + imatinib-, and radioimmunotherapy-treated tumors, respectively. A continuous 14-day irradiation of imatinib-treated xenografts reduces levels of PDGFr-β and phosphorylated PDGFr-β by 5.3× and 4×, compared to earlier times. Human PDGF-B is upregulated suggesting that the survival signaling via the autocrine pathway is also triggered after stromal injury. These findings indicate that therapies targeting pancreatic cancer stromal components may have unintended mitogenic effects and that these effects can be reversed when imatinib is used in conjunction with radioimmunotherapy.

Radioiodinated Agents for Imaging Multidrug Resistant Tumors

Diagnostic agents enabling characterization of multidrug resistance (MDR) in tumors can aid in the selection of chemotherapy regimens. We report here synthesis and evaluation of radiopharmaceuticals based on the second-generation MDR-reversing drug MS-209. 5-[3-{4-(2-Phenyl-2-(4-[(125)I]iodo-phenyl)acetyl)piperazin-1-yl}-2-hydroxypropoxy]quino-line (17) was prepared from the 4-tributylstannyl precursor (16) in >95% radiochemical yield. (16) was synthesized in a six-step process with the overall yield of 25%. In vitro studies were conducted in MES-SA (drug-sensitive) and MES-SA/Dx5 (MDR) human uterine sarcoma cell lines. In vivo studies were performed in athymic mice bearing MES-SA and MES-SA/Dx5 xenografts. The uptake of (17) is higher in MES-SA than MES-SA/Dx5 cells. The uptake and efflux of (17) depend on temperature and concentration, and indicate active transport mechanism(s). Incubation of drug sensitive MES-SA cells with verapamil or (15), a nonradioactive analog of (17), alters the cellular retention of radioactivity only marginally. However, MES-SA/Dx5 cells retain approximately 12% more of (17) when incubated with 10 muM verapamil. The addition of (15) or high concentrations of (17) also increase the uptake of (17) in MES-SA/Dx5 up to 200%, depending on the concentration and temperature. The dependence of (17) uptake on the MDR status is also evident in the ex vivo binding studies. In vivo tests in mice xenografted simultaneously with both tumor cell lines indicate distinct pharmacokinetics for each tumor. The absorption half-life in MES-SA/Dx5 xenograft is approximately 10x shorter and the mean residence time approximately 50% shorter compared to MES-SA xenograft in the same mouse. Radioiodinated derivatives of MS-209 appear to be good indicators of multidrug resistance.

Preclinical evaluation of investigational radiopharmaceutical RISAD-P intended for use as a diagnostic and molecular radiotherapy agent for prostate cancer.

The androgen receptor (AR) plays a dominant role in the pathogenesis of prostate cancer. 5‐Radioiodo‐3′‐O‐(17β‐succinyl‐5α‐androstan‐3‐one)‐2′‐deoxyuridin‐5′‐yl phosphate (RISAD‐P) is an AR‐targeting reagent developed for noninvasive assessment of AR and proliferative status of the AR‐expressing tumors, and for molecular radiotherapy with Auger electron‐emitting radionuclides. In this study, the preclinical toxicity and targeting potential of RISAD‐P was evaluated.

Radiosynthesis of microtubule-targeted theranostic methyl N-[5-(3’-radiohalobenzoyl)-1H-benzimidazol-2-yl]carbamates

Microtubules are a target for a broad spectrum of drugs used as chemotherapeutics to treat hematological malignancies and solid tumors. Most of these drugs have significant dose-limiting toxicities including peripheral neuropathies that can be debilitating and permanent. In an ongoing effort to develop safer and more effective drugs, benzimidazole-based compounds are being developed as replacement for vincristine and similar agents. In this report, we describe radiosyntheses of novel microtubule-targeting methyl N-[5-(3-radiohalobenzoyl)-1H-benzimidazol-2-yl]carbamates 4 that are intended as potential imaging agents and molecular radiotherapeutics. 125 I- and 131 I-radiolabeled derivatives were prepared either by direct radioiodination of methyl N-(6-benzoyl-1H-benzimidazol-2-yl)carbamate 1 or radioiododestannylation of the corresponding stannane precursor 3. The direct radioiodination was conducted in a solution of 1 in triflic acid and produced after ~1xa0hour at elevated temperatures and HPLC purification on average 62% of the no-carrier added products 125 I-4 and 131 I-4. Radioiododestannylation of 3-trimethylstannane 3 proceeded with ease at room temperature in the presence of H2 O2 as the oxidant and produced no-carrier-added 125 I-4 and 131 I-4 in high isolated yields, on average 85%. The radiohalodestannylation protocol is universal and can be applied to other radiohalides including 124 I to produce 124 I-4, a positron emission tomography agent, and 211 At to produce 211 At-4, an α-particle emitting radiotherapeutic.