Different radiolabelling methods alter the pharmacokinetic and biodistribution properties of Plasminogen Activator Inhibitor Type 2 (PAI-2) forms

Introduction: Tumour-associated urokinase plasminogen activator (uPA) is a critical marker of invasion and metastasis, and it is recognised as having strong prognostic relevance as well as being a therapeutic target. The specific uPA inhibitor plasminogen activator inhibitor type-2 (PAI-2, SerpinB2) specifically targets cell bound uPA and is internalised. Furthermore, preclinical studies have established the “proof-of-principle” of uPA-targeting by PAI-2-cytotoxin conjugates in human carcinoma models. However, these studies also suggest that PAI-2 is rapidly cleared via the renal system with low total dose reaching the tumour. In this study, a comparative single photon emission computed tomography (SPECT) and biodistribution (BD) analysis of different forms of PAI-2 labelled with the radioisotopes iodine-123 (123I) and technetium-99m (99mTc) was undertaken. Methods: The pharmacokinetic (PK) properties and BD of wild-type, ΔCD-loop and PEGylated ΔCD-loop PAI-2 labelled with the commonly used diagnostic SPECT radioisotopes 99mTc or 123I were compared in mouse models of human prostate carcinoma. Whole body SPECT imaging was also performed. Results: Both wild-type or the shorter but active ΔCD-loop form of PAI-2 123I-labelled indirectly via conjugation to free amine groups (termed 123I-Bn-PAI-2) exhibited low tumour uptake, rapid excretion and similar PK profiles. Preliminary studies with a short branched-chain PEGylated 123I-Bn-PAI-2 ΔCD-loop indicated an increase in blood retention time and tumour uptake. All 123I-Bn-labelled radiotracers were largely excreted through the kidneys. By comparison, both wild-type 123I-PAI-2 (labelled directly via tyrosine residues) and 99mTc-PAI-2 displayed different PK/BD patterns compared to 123IBn- PAI-2, suggesting greater liver based catabolism and thus slower elimination. SPECT imaging mimicked the BD results of all radiotracers. Conclusion: The different labelling methods gave distinct PAI-2 BD and tumour uptake profiles, with radioiodination resulting in the best non-tumour organ clearance profiles. Preliminary analyses with short branched-chain PEGylated 123I-Bn-PAI-2 ΔCD-loop suggest that further investigations with other PEGylation reagents are required to optimise this approach for tumour imaging. These findings impact on the use of PAI-2 for drug delivery and/or diagnostic development

Special Libraries, May-June 1954

Volume 45, Issue 5https://scholarworks.sjsu.edu/sla_sl_1954/1004/thumbnail.jp

The CD-loop of PAI-2 (SERPINB2) is redundant in the targeting, inhibition and clearance of cell surface uPA activity

<p>Abstract</p> <p>Background</p> <p>Plasminogen activator inhibitor type-2 (PAI-2, SERPINB2) is an irreversible, specific inhibitor of the urokinase plasminogen activator (uPA). Since overexpression of uPA at the surface of cancer cells is linked to malignancy, targeting of uPA by exogenous recombinant PAI-2 has been proposed as the basis of potential cancer therapies. To this end, reproducible yields of high purity protein that maintains this targeting ability is required. Herein we validate the use <it>in vitro </it>of recombinant 6 × His-tagged-PAI-2 lacking the intrahelical loop between C and D alpha-helices (PAI-2 ΔCD-loop) for these purposes.</p> <p>Results</p> <p>We show that PAI-2 ΔCD-loop expressed and purified from the pQE9 vector system presents an easier purification target than the previously used pET15b system. Additionally, PAI-2 ΔCD-loop gave both higher yield and purity than wild-type PAI-2 expressed and purified under identical conditions. Importantly, absence of the CD-loop had no impact on the inhibition of both solution phase and cell surface uPA or on the clearance of receptor bound uPA from the cell surface. Furthermore, uPA:PAI-2 ΔCD-loop complexes had similar binding kinetics (K_D~5 nM) with the endocytosis receptor Very Low Density Lipoprotein Receptor (VLDLR) to that previously published for uPA:PAI-2 complexes.</p> <p>Conclusion</p> <p>We demonstrate that the CD-loop is redundant for the purposes of cellular uPA inhibition and cell surface clearance (endocytosis) and is thus suitable for the development of anti-uPA targeted cancer therapeutics.</p

Preclinical Evaluation of Novel All-in-one Formulations of 5-Fluorouracil and Folinic Acid with Reduced Toxicity Profiles - Supplementary Data

Objectives: 5-Fluorouracil (5-FU) in combination with its synergistic biomodulator folinic acid maintains a pivotal position in cancer chemotherapy. However, clinical limitations persist with the administration of these drugs in combination including phlebitis and catheter blockages, which are associated with reduced efficacy and/or quality of life for patients. We have previously reported novel all-in-one, pH neutral, parenteral 5-FU and folinic acid formulations (termed Fluorodex) incorporating beta-cyclodextrins. Fluorodex maintains potency while overcoming the accepted incompatibility of 5-FU and folinic acid.Methods: We performed toxicological, pharmacokinetic and biodistribution, and efficacy evaluations of Fluorodex compared to 5-FU:folinic acid using several administration routes and schedules in two rodent models. These were compared to dose-matched sequential administration of 5-FU:folinic acid.Results: Fluorodex showed bioequivalence to 5-FU:folinic acid as assessed by tissue distribution and pharmacokinetics of 5-FU, but was generally better tolerated as determined by weight loss, hematological and other clinical parameters. Compared to 5-FU:folinic acid, Fluorodex was also associated with reduced phlebitis using a rabbit ear vein model. Furthermore, equivalent to enhanced efficacy of Fluorodex compared to 5-FU:folinic acid against human carcinoma tumour models in mice was observed.Conclusions: These novel all-in-one formulations represent a superior injectable form of 5- FU that allows co-delivery of folinic acid. This should translate to improved patient tolerability with potential for enhanced efficacy

Towards clinical translation of ligand-functionalized liposomes in targeted cancer therapy: challenges and opportunities

The development of therapeutic resistance to targeted anticancer therapies remains a significant clinical problem, with intratumoral heterogeneity playing a key role. In this context, improving the therapeutic outcome through simultaneous targeting of multiple tumor cell subtypes within a heterogeneous tumor is a promising approach. Liposomes have emerged as useful drug carriers that can reduce systemic toxicity and increase drug delivery to the tumor site. While clinically-used liposomal drug formulations show marked therapeutic advantages over free drug formulations, ligand-functionalized liposome drug formulations that can target multiple tumor cell subtypes may further improve the therapeutic efficacy by facilitating drug delivery to a broader population of tumor cells making up the heterogeneous tumor tissue. Ligand-directed liposomes enable the so-called active targeting of cell receptors via surface-attached ligands that direct drug uptake into tumor cells or tumor-associated stromal cells, and so can increase the selectivity of drug delivery. Despite promising preclinical results demonstrating improved targeting and anti-tumor effects of ligand-directed liposomes, there has been limited translation of this approach to the clinic. Key challenges for translation include the lack of established methods to scale up production and comprehensively characterize ligand-functionalized liposome formulations, and the inadequate recapitulation of in vivo tumors in the preclinical models currently used to evaluate their performance. Herein, we discuss the utility of recent ligand-directed liposome approaches, with a focus on dual-ligand liposomes, for the treatment of solid tumors and examine the drawbacks limiting their progression to clinical adoption

Establishment of novel long-term cultures from EpCAM positive and negative circulating tumour cells from patients with metastatic gastroesophageal cancer

Circulating tumour cell (CTC) enumeration and profiling has been established as a valuable clinical tool in many solid malignancies. A key challenge in CTC research is the limited number of cells available for study. Ex vivo CTC culture permits expansion of these rare cell populations for detailed characterisation, functional assays including drug sensitivity testing, and investigation of the pathobiology of metastases. We report for the first time the establishment and characterisation of two continuous CTC lines from patients with gastroesophageal cancer. The two cell lines (designated UWG01CTC and UWG02CTC) demonstrated rapid tumorigenic growth in immunodeficient mice and exhibit distinct genotypic and phenotypic profiles which are consistent with the tumours of origin. UWG02CTC exhibits an EpCAM+, cytokeratin+, CD44+ phenotype, while UWG01CTC, which was derived from a patient with metastatic neuroendocrine cancer, displays an EpCAM−, weak cytokeratin phenotype, with strong expression of neuroendocrine markers. Further, the two cell lines show distinct differences in drug and radiation sensitivity which match differential cancer-associated gene expression pathways. This is strong evidence implicating EpCAM negative CTCs in metastasis. These novel, well characterised, long-term CTC cell lines from gastroesophageal cancer will facilitate ongoing research into metastasis and the discovery of therapeutic targets

Examining the efficacy of localised gemcitabine therapy for the treatment of pancreatic cancer using a hybrid agent-based model

The prognosis for pancreatic ductal adenocarcinoma (PDAC) patients has not significantly improved in the past 3 decades, highlighting the need for more effective treatment approaches. Poor patient outcomes and lack of response to therapy can be attributed, in part, to a lack of uptake of perfusion of systemically administered chemotherapeutic drugs into the tumour. Wet-spun alginate fibres loaded with the chemotherapeutic agent gemcitabine have been developed as a potential tool for overcoming the barriers in delivery of systemically administrated drugs to the PDAC tumour microenvironment by delivering high concentrations of drug to the tumour directly over an extended period. While exciting, the practicality, safety, and effectiveness of these devices in a clinical setting requires further investigation. Furthermore, an in-depth assessment of the drug-release rate from these devices needs to be undertaken to determine whether an optimal release profile exists. Using a hybrid computational model (agent-based model and partial differential equation system), we developed a simulation of pancreatic tumour growth and response to treatment with gemcitabine loaded alginate fibres. The model was calibrated using in vitro and in vivo data and simulated using a finite volume method discretisation. We then used the model to compare different intratumoural implantation protocols and gemcitabine-release rates. In our model, the primary driver of pancreatic tumour growth was the rate of tumour cell division. We were able to demonstrate that intratumoural placement of gemcitabine loaded fibres was more effective than peritumoural placement. Additionally, we quantified the efficacy of different release profiles from the implanted fibres that have not yet been tested experimentally. Altogether, the model developed here is a tool that can be used to investigate other drug delivery devices to improve the arsenal of treatments available for PDAC and other difficult-to-treat cancers in the future

N-alkyl-substituted isatins enhance P2X7 receptor-induced interleukin-1β release from murine macrophages

Extracellular adenosine 5′-triphosphate (ATP) activates the P2X7 receptor channel to induce the rapid release of the proinflammatory cytokine, interleukin- (IL-) 1β, from macrophages. Microtubule rearrangements are thought to be involved in this process. Some isatin derivatives alter microtubules and display anticancer activities. The current study investigated the effect of isatin and seven structurally diverse isatin derivatives on P2X7-mediated IL-1β release from murine J774 macrophages. ATP-induced IL-1β and lactate dehydrogenase (LDH) release were assessed by specific colorimetric assays. P2X7 activity was determined by flow cytometric measurements of ATP-induced cation dye uptake. Cytotoxicity of isatin derivatives was determined using a tetrazolium-based colorimetric assay. ATP caused rapid IL-1β release in a concentration-dependent manner, and this process was completely impaired by the P2X7 antagonist, AZ10606120. In contrast, 5,7-dibromo-N-(p-methoxybenzyl)isatin (NAI) and 3-4-[5,7-dibromo-1-(4-methoxybenzyl)-2-oxoindolin-3-ylidenamino]phenylpropanoic acid (NAI-imine) enhanced P2X7-induced IL-1β release by twofold compared to that of isatin and the parent molecule, 5,7-dibromoisatin. NAI and NAI-imine had minimal effect on P2X7-induced dye uptake and LDH release. In contrast, 24-hour incubation with NAI and NAI-imine (in the absence of exogenous ATP) induced macrophage death in a concentration-dependent manner. In conclusion, this study demonstrates that N-alkyl-substituted isatins enhance P2X7 receptor-induced IL-1β release from murine macrophages. Thus, in addition to direct anticancer effects, these compounds may also impact inflammatory and immune cells within the tumor microenvironment

N-alkylisatin-loaded liposomes target the urokinase plasminogen activator system in breast cancer

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. The urokinase plasminogen activator and its receptor (uPA/uPAR) are biomarkers for metastasis, especially in triple-negative breast cancer. We prepared anti-mitotic N-alkylisatin (N-AI)-loaded liposomes functionalized with the uPA/uPAR targeting ligand, plasminogen activator inhibitor type 2 (PAI-2/SerpinB2), and assessed liposome uptake in vitro and in vivo. Receptor-dependent uptake of PAI-2-functionalized liposomes was significantly higher in the uPA/uPAR overexpressing MDA-MB-231 breast cancer cell line relative to the low uPAR/uPAR expressing MCF-7 breast cancer cell line. Furthermore, N-AI cytotoxicity was enhanced in a receptor-dependent manner. In vivo, PAI-2 N-AI liposomes had a plasma half-life of 5.82 h and showed an increased accumulation at the primary tumor site in an orthotopic MDA-MB-231 BALB/c-Fox1nu/Ausb xenograft mouse model, relative to the non-functionalized liposomes, up to 6 h post-injection. These findings support the further development of N-AI-loaded PAI-2-functionalized liposomes for uPA/uPAR-positive breast cancer, especially against triple-negative breast cancer, for which the prognosis is poor and treatment is limited

Cancer biology: molecular and genetic basis

Cancer is a disease of uncontrolled growth and proliferation whereby cells have escaped the body\u27s normal growth control mechanisms and have gained the ability to divide indefinitely. It is a multi-step process that requires the accumulation of many genetic changes over time (Figure 1). These genetic alterations involve activation of proto-oncogenes to oncogenes, deregulation of tumour suppressor genes and DNA repair genes and \u27immortalisation\u27 which will be discussed in this chapter