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.

Post-Transplant Cyclophosphamide Combined with Brilliant Blue G Reduces Graft-versus-Host Disease without Compromising Graft-versus-Leukaemia Immunity in Humanised Mice.

Allogeneic haematopoietic stem cell transplantation (HSCT) leads to the establishment of graft-versus-leukaemia (GVL) immunity, but in many cases also results in the development of graft-versus-host disease (GVHD). This study aimed to determine if P2X7 antagonism using Brilliant Blue G (BBG) could improve the beneficial effects of post-transplant cyclophosphamide (PTCy) in a humanised mouse model of GVHD, without comprising GVL immunity. NOD.Cg-Prkdcscid Il2rgtm1Wjl (NSG) mice were injected with human peripheral blood mononuclear cells (PBMCs) (Day 0), then with cyclophosphamide (33 mg/kg) on Days 3 and 4, and with BBG (50 mg/kg) (or saline) on Days 0-10. PTCy with BBG reduced clinical GVHD development like that of PTCy alone. However, histological analysis revealed that the combined treatment reduced liver GVHD to a greater extent than PTCy alone. Flow cytometric analyses revealed that this reduction in liver GVHD by PTCy with BBG corresponded to an increase in human splenic CD39+ Tregs and a decrease in human serum interferon-γ concentrations. In additional experiments, humanised NSG mice, following combined treatment, were injected with human THP-1 acute myeloid leukaemia cells on Day 14. Flow cytometric analyses of liver CD33+ THP-1 cells showed that PTCy with BBG did not mitigate GVL immunity. In summary, PTCy combined with BBG can reduce GVHD without compromising GVL immunity. Future studies investigating P2X7 antagonism in combination with PTCy may lead to the development of novel treatments that more effectively reduce GVHD in allogeneic HSCT patients without promoting leukaemia relapse.

Recent Advances in Targeting the Urokinase Plasminogen Activator with Nanotherapeutics.

The aberrant proteolytic landscape of the tumor microenvironment is a key contributor of cancer progression. Overexpression of urokinase plasminogen activator (uPA) and/or its associated cell-surface receptor (uPAR) in tumor versus normal tissue is significantly associated with worse clinicopathological features and poorer patient survival across multiple cancer types. This is linked to mechanisms that facilitate tumor cell invasion and migration, via direct and downstream activation of various proteolytic processes that degrade the extracellular matrix─ultimately leading to metastasis. Targeting uPA has thus long been considered an attractive anticancer strategy. However, poor bioavailability of several uPA-selective small-molecule inhibitors has limited early clinical progress. Nanodelivery systems have emerged as an exciting method to enhance the pharmacokinetic (PK) profile of existing chemotherapeutics, allowing increased circulation time, improved bioavailability, and targeted delivery to tumor tissue. Combining uPA inhibitors with nanoparticle-based delivery systems thus offers a remarkable opportunity to overcome existing PK challenges associated with conventional uPA inhibitors, while leveraging potent candidates into novel targeted nanotherapeutics for an improved anticancer response in uPA positive tumors.

Non-Viral Vector-Mediated Gene Therapy for ALS: Challenges and Future Perspectives.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease, for which no effective treatment is yet available to either slow or terminate it. Recent advances in gene therapy renew hope for developing an effective approach to control this disease. Non-viral vectors, such as lipid- and polymer-based nanoparticles, cationic polymers, and exosomes, can effectively transfer genes into primary neurons. The resulting gene expression can be long-term, stable, and without immunological complications, which is essential for the effective management of neurological disorders. This Review will first describe the current research and clinical stage of novel therapies for ALS. It will then touch on the journey of non-viral vector use in ALS, subsequently highlighting the application of non-viral vector-mediated gene therapy. The bottlenecks in the translation of non-viral vectors for ALS treatment are also discussed, including the biological barriers of systemic administration and the issues of "when, where, and how much?" for effective gene delivery. The prospect of employing emerging techniques, such as CRISPR-Cas9 gene editing, stem cell methodology, and low-intensity focused ultrasound for fueling the transport of non-viral vectors to the central nervous system for personalized gene therapy, is briefly discussed in the context of ALS. Despite the challenging road that lies ahead, with the current expansion in interest and technological advancement in non-viral vector-delivered gene therapy for ALS, we hold hope that the field is headed toward a positive future.

SOD1A4V aggregation alters ubiquitin homeostasis in a cell model of ALS.

A hallmark of amyotrophic lateral sclerosis (ALS) pathology is the accumulation of ubiquitylated protein inclusions within motor neurons. Recent studies suggest the sequestration of ubiquitin (Ub) into inclusions reduces the availability of free Ub, which is essential for cellular function and survival. However, the dynamics of the Ub landscape in ALS have not yet been described. Here, we show that Ub homeostasis is altered in a cell model of ALS induced by expressing mutant SOD1 (SOD1A4V). By monitoring the distribution of Ub in cells expressing SOD1A4V, we show that Ub is present at the earliest stages of SOD1A4V aggregation, and that cells containing SOD1A4V aggregates have greater ubiquitin-proteasome system (UPS) dysfunction. Furthermore, SOD1A4V aggregation is associated with the redistribution of Ub and depletion of the free Ub pool. Ubiquitomics analysis indicates that expression of SOD1A4V is associated with a shift of Ub to a pool of supersaturated proteins, including those associated with oxidative phosphorylation and metabolism, corresponding with altered mitochondrial morphology and function. Taken together, these results suggest that misfolded SOD1 contributes to UPS dysfunction and that Ub homeostasis is an important target for monitoring pathological changes in ALS.This article has an associated First Person interview with the first author of the paper.

N-alkylisatin-based microtubule destabilizers bind to the colchicine site on tubulin and retain efficacy in drug resistant acute lymphoblastic leukemia cell lines with less in vitro neurotoxicity.

BACKGROUND: Drug resistance and chemotherapy-induced peripheral neuropathy continue to be significant problems in the successful treatment of acute lymphoblastic leukemia (ALL). 5,7-Dibromo-N-alkylisatins, a class of potent microtubule destabilizers, are a promising alternative to traditionally used antimitotics with previous demonstrated efficacy against solid tumours in vivo and ability to overcome P-glycoprotein (P-gp) mediated drug resistance in lymphoma and sarcoma cell lines in vitro. In this study, three di-brominated N-alkylisatins were assessed for their ability to retain potency in vincristine (VCR) and 2-methoxyestradiol (2ME2) resistant ALL cell lines. For the first time, in vitro neurotoxicity was also investigated in order to establish their suitability as candidate drugs for future use in ALL treatment. METHODS: Vincristine resistant (CEM-VCR R) and 2-methoxyestradiol resistant (CEM/2ME2-28.8R) ALL cell lines were used to investigate the ability of N-alkylisatins to overcome chemoresistance. Interaction of N-alkylisatins with tubulin at the the colchicine-binding site was studied by competitive assay using the fluorescent colchicine analogue MTC. Human neuroblastoma SH-SY5Y cells differentiated into a morphological and functional dopaminergic-like neurotransmitter phenotype were used for neurotoxicity and neurofunctional assays. Two-way ANOVA followed by a Tukey's post hoc test or a two-tailed paired t test was used to determine statistical significance. RESULTS: CEM-VCR R and CEM/2ME2-28.8R cells displayed resistance indices of > 100 to VCR and 2-ME2, respectively. CEM-VCR R cells additionally displayed a multi-drug resistant phenotype with significant cross resistance to vinblastine, 2ME2, colchicine and paclitaxel consistent with P-gp overexpression. Despite differences in resistance mechanisms observed between the two cell lines, the N-alkylisatins displayed bioequivalent dose-dependent cytotoxicity to that of the parental control cell line. The N-alkylisatins proved to be significantly less neurotoxic towards differentiated SH-SY5Y cells than VCR and vinblastine, evidenced by increased neurite length and number of neurite branch points. Neuronal cells treated with 5,7-dibromo-N-(p-hydroxymethylbenzyl)isatin showed significantly higher voltage-gated sodium channel function than those treated with Vinca alkaloids, strongly supportive of continued action potential firing. CONCLUSIONS: The N-alkylisatins are able to retain cytotoxicity towards ALL cell lines with functionally distinct drug resistance mechanisms and show potential for reduced neurotoxicity. As such they pose as promising candidates for future implementation into anticancer regimes for ALL. Further in vivo studies are therefore warranted.

Preparation and in vitro assessment of wet-spun gemcitabine-loaded polymeric fibers: Towards localized drug delivery for the treatment of pancreatic cancer.

BACKGROUND/OBJECTIVES: There has been minimal improvement in the prognosis of pancreatic cancer cases in the past 3 decades highlighting the crucial need for more effective therapeutic approaches. A drug delivery system capable of locally delivering high concentrations of chemotherapeutics directly at the site of the tumor is clearly required. The aim of this study was to fabricate and characterize the biophysical properties of gemcitabine-eluting wet-spun polymeric fibers for localized drug delivery applications. METHODS/RESULTS: Fibers spun from alginate or chitosan solutions with or without the anticancer drug gemcitabine had a uniform surface area, were internally homogeneous and ranged from 50-120 µm in diameter. Drug encapsulation ranged from 13-52%, depending on the type and concentration of polymer used. Gemcitabine displayed first-order release kinetics where 64-82% of the loaded drug was rapidly released within the first 10 h followed by a sustained release over the next 134 h. A time dependent inhibition of ex vivo tumor spheroid growth and cell viability was observed after incubation with gemcitabine-loaded fibers but not control fibers. CONCLUSION: With further development these studies could lead to the manufacture of a safe and effective delivery system designed to combat non-resectable pancreatic cancer for which currently there is minimal chance of cure.

Translationally controlled tumour protein TCTP is induced early in human colorectal tumours and contributes to the resistance of HCT116 colon cancer cells to 5-FU and oxaliplatin.

BACKGROUND: Translationally controlled tumour protein TCTP is an anti-apoptotic protein frequently overexpressed in cancers, where high levels are often associated with poor patient outcome. TCTP may be involved in protecting cancer cells against the cytotoxic action of anti-cancer drugs. Here we study the early increase of TCTP levels in human colorectal cancer (CRC) and the regulation of TCTP expression in HCT116 colon cancer cells, in response to treatment with the anti-cancer drugs 5-FU and oxaliplatin. METHODS: Using immunohistochemistry, we assessed TCTP levels in surgical samples from adenomas and adenocarcinomas of the colon, compared to normal colon tissue. We also studied the regulation of TCTP in HCT116 colon cancer cells in response to 5-FU and oxaliplatin by western blotting. TCTP mRNA levels were assessed by RT-qPCR. We used mTOR kinase inhibitors to demonstrate mTOR-dependent translational regulation of TCTP under these conditions. Employing the Real-Time Cell Analysis (RTCA) System and the MTS assay, we investigated the effect of TCTP-knockdown on the sensitivity of HCT116 cells to the anti-cancer drugs 5-FU and oxaliplatin. RESULTS: 1. TCTP levels are significantly increased in colon adenomas and adenocarcinomas, compared to normal colon tissue. 2. TCTP protein levels are about 4-fold upregulated in HCT116 colon cancer cells, in response to 5-FU and oxaliplatin treatment, whereas TCTP mRNA levels are down regulated. 3. mTOR kinase inhibitors prevented the up-regulation of TCTP protein, indicating that TCTP is translationally regulated through the mTOR complex 1 signalling pathway under these conditions. 4. Using two cellular assay systems, we demonstrated that TCTP-knockdown sensitises HCT116 cells to the cytotoxicity caused by 5-FU and oxaliplatin. CONCLUSIONS: Our results demonstrate that TCTP levels increase significantly in the early stages of CRC development. In colon cancer cells, expression of this protein is largely upregulated during treatment with the DNA-damaging anti-cancer drugs 5-FU and oxaliplatin, as part of the cellular stress response. TCTP may thus contribute to the development of anti-cancer drug resistance. These findings indicate that TCTP might be suitable as a biomarker and that combinatorial treatment using 5-FU/oxaliplatin, together with mTOR kinase inhibitors, could be a route to preventing the development of resistance to these drugs.

Probing the pharmacokinetics of cucurbit[7, 8 and 10]uril: and a dinuclear ruthenium antimicrobial complex encapsulated in cucurbit[10]uril.

The relatively non-toxic family of cucurbit[n]uril, Q[n], have shown considerable potential in vitro as drug delivery agents, with only a few examples of pharmacokinetic (PK) studies for drug⊂Q[n]. Drug-free Q[n] PK studies are the next step in determining the pharmacological applicability in their drug delivery potential. The results for the first PK and bio-distribution of drug-free 14C-Q[7] are described for administration via intravenous (i.v.) and intraperitoneal (i.p.) dosing. A study of oral administration of drug-free 14C-Q[8] has also been undertaken to determine the time course for the gastrointestinal tract (GIT), absorption and subsequent bio-distribution. Q[10], a potential drug carrier for larger drugs, was evaluated for its effect on the PK profile of a dinuclear ruthenium complex (Rubb12), a potential antimicrobial agent. The Rubb12⊂Q[10] complex and free Rubb12 were administered by i.v. to determine differences in Rubb12 plasma concentrations and organ accumulation. Interestingly, the PK profiles and bio-distribution observed for Q[7] showed similarities to those of Rubb12⊂Q[10]. Drug-free Q[7] has a relatively fast plasma clearance and a generally low organ accumulation except for the kidneys. Drug-free Q[8] showed a low absorption from the GIT into the blood stream but the small percentage absorbed reflected the organ accumulation of Q[7]. These results provide a better understanding of the probable PK profile and bio-distribution for a drug⊂Q[n] through the influence of the drug delivery vehicle and the positive clearance of drug-free Q[n] via the kidneys supports its potential value in future drug delivery applications.

N-Alkyl-Substituted Isatins Enhance P2X7 Receptor-Induced Interleukin-1ß 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-) 1ß, 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-1ß release from murine J774 macrophages. ATP-induced IL-1ß 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-1ß 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]phenyl}propanoic acid (NAI-imine) enhanced P2X7-induced IL-1ß 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-1ß release from murine macrophages. Thus, in addition to direct anticancer effects, these compounds may also impact inflammatory and immune cells within the tumor microenvironment.

Graft-versus-leukaemia immunity is retained following treatment with post-transplant cyclophosphamide alone or combined with tocilizumab in humanised mice.

Objectives: Donor haematopoietic stem cell transplantation treats leukaemia by inducing graft-versus-leukaemia (GVL) immunity. However, this benefit is often mitigated by graft-versus-host disease (GVHD), which is reduced by post-transplant cyclophosphamide (PTCy) alone or combined with tocilizumab (TOC) in humanised mice. This study established a preclinical humanised mouse model of GVL and investigated whether PTCy alone or combined with TOC impacts GVL immunity. Methods: NOD-scid-IL2Rγnull mice were injected with 2 × 107 human peripheral blood mononuclear cells (hPBMCs) on day 0 and with 1 × 106 THP-1 acute myeloid leukaemia cells on day 14. In subsequent experiments, mice were also injected with PTCy (33 mg kg-1) or Dulbecco's phosphate buffered saline (PBS) on days 3 and 4, alone or combined with TOC or control antibody (25 mg kg-1) twice weekly for 28 days. Clinical signs of disease were monitored until day 42. Results: Mice with hPBMCs from three different donors and THP-1 cells showed similar survival, clinical score and weight loss. hCD33+ leukaemia cells were minimal in mice reconstituted with hPBMCs from two donors but present in mice with hPBMCs from a third donor, suggesting donor-specific GVL responses. hPBMC-injected mice treated with PTCy alone or combined with TOC (PTCy + TOC) demonstrated prolonged survival compared to control mice. PTCy alone and PTCy + TOC-treated mice with hPBMCs showed minimal hepatic hCD33+ leukaemia cells, indicating sustained GVL immunity. Further, the combination of PTCy + TOC reduced histological damage in the lung and liver. Conclusion: Collectively, this research demonstrates that PTCy alone or combined with TOC impairs GVHD without compromising GVL immunity.

The antiangiogenic properties of sulfated ß-cyclodextrins in anticancer formulations incorporating 5-fluorouracil.

Sulfated ß-cyclodextrins (S-ß-CDs) are useful excipients for improving the solubility of drugs. One such formulation incorporating 5-fluorouracil (5-FU), termed FD(S), showed improved efficacy over 5-FU alone in orthotopic carcinoma xenograft models. S-ß-CDs have heparin-like anticoagulant properties, which may have contributed toward the improved antitumor effect of FD(S). S-ß-CDs have also been reported to modify a number of processes involved in angiogenesis. Although the anticoagulant nature of S-ß-CDs was established, the antiangiogenic properties of S-ß-CDs within FD(S) were unknown. The effect of S-ß-CD and FD(S) on the proliferation and migration of endothelial cells in live-cell kinetic assays, and the reorganization of human umbilical vein endothelial cells into tubule structures in vitro was assessed. The effects of S-ß-CD on angiogenesis in vitro were validated ex vivo using the rat aorta ring assay and the chick embryo chorioallantoic membrane assay. S-ß-CD does not alter proliferative endothelial cell sensitivity to 5-FU cytotoxicity. S-ß-CD alone and within FD(S) significantly inhibited angiogenesis by impeding endothelial cell migration, resulting in the inhibition of tubule formation and hence new vasculature. In addition to the cytotoxic action of the drug 5-FU, therapeutic inhibition of angiogenesis by S-ß-CDs within FD(S) could potentially limit local invasion and metastases. This has important implications for the exploitation of S-ß-CDs for drug formulation improvements or for drug delivery of anticancer biologics.

Localized delivery of immunotherapy via implantable scaffolds for breast cancer treatment.

Breast cancer remains a leading global cause of morbidity and mortality. While the field of immunotherapy is a promising avenue of investigation and has revolutionized the standard of care for melanoma and lung cancer, modest response rates and a high incidence of immune-related adverse events often necessitate the administration of a sub-therapeutic dose or treatment cessation. Injectable and implantable drug delivery devices present a novel strategy to achieve sustained delivery of potent concentrations of drug directly to the tumor site and minimize systemic toxicity. This review will address the current limitations with conventional immunotherapy for breast cancer treatment, and the recent developments and future prospects in localized delivery strategies. We describe implantable scaffolds and injectable biomaterials for the localized delivery of immunotherapy, which can improve the safety and efficacy of immunotherapies. We discuss the limitations of these delivery systems, such as the influence of shape and material type on drug release and tumor uptake. The challenges of clinical translation, such as the availability of appropriate preclinical animal models and accurate reporting are also discussed. Considerations of these issues will pave the way for effective new therapies that will improve treatment response, patient survival and quality of life for breast cancer patients.

Preclinical evaluation of novel, all-in-one formulations of 5-fluorouracil and folinic acid with reduced toxicity profiles.

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

Synthesis and hydrolytic evaluation of acid-labile imine-linked cytotoxic isatin model systems.

In this study a series of isatin-based, pH-sensitive aryl imine derivatives with differing aromatic substituents and substitution patterns were synthesised and their acid-catalysed hydrolysis evaluated. These derivatives were functionalised at the C3 carbonyl group of a potent N-substituted isatin cytotoxin and were stable at physiological pH but readily cleaved at pH 4.5. Observed rates of hydrolysis for the embedded imine-acid moiety were in the order para-phenylpropionic acid>phenylacetic acid (para>meta)>benzoic acid (meta>para). The ability to fine-tune hydrolysis rates in this way has potential implications for optimising imine linked, tumour targeting cytotoxin-protein conjugates.

3D-Printed Coaxial Hydrogel Patches with Mussel-Inspired Elements for Prolonged Release of Gemcitabine.

With the aim of fabricating drug-loaded implantable patches, a 3D printing technique was employed to produce novel coaxial hydrogel patches. The core-section of these patches contained a dopamine-modified methacrylated alginate hydrogel loaded with a chemotherapeutic drug (Gemcitabine), while their shell section was solely comprised of a methacrylated alginate hydrogel. Subsequently, these patches were further modified with CaCO3 cross linker and a polylactic acid (PLA) coating to facilitate prolonged release of the drug. Consequently, the results showed that addition of CaCO3 to the formula enhanced the mechanical properties of the patches and significantly reduced their swelling ratio as compared to that for patches without CaCO3. Furthermore, addition of PLA coating to CaCO3-containing patches has further reduced their swelling ratio, which then significantly slowed down the release of Gemcitabine, to a point where 4-layered patches could release the drug over a period of 7 days in vitro. Remarkably, it was shown that 3-layered and 4-layered Gemcitabine loaded patches were successful in inhibiting pancreatic cancer cell growth for a period of 14 days when tested in vitro. Lastly, in vivo experiments showed that gemcitabine-loaded 4-layered patches were capable of reducing the tumor growth rate and caused no severe toxicity when tested in mice. Altogether, 3D printed hydrogel patches might be used as biocompatible implants for local delivery of drugs to diseased site, to either shrink the tumor or to prevent the tumor recurrence after resection.

CuATSM improves motor function and extends survival but is not tolerated at a high dose in SOD1G93A mice with a C57BL/6 background.

The synthetic copper-containing compound, CuATSM, has emerged as one of the most promising drug candidates developed for the treatment of amyotrophic lateral sclerosis (ALS). Multiple studies have reported CuATSM treatment provides therapeutic efficacy in various mouse models of ALS without any observable adverse effects. Moreover, recent results from an open label clinical study suggested that daily oral dosing with CuATSM slows disease progression in patients with both sporadic and familial ALS, providing encouraging support for CuATSM in the treatment of ALS. Here, we assessed CuATSM in high copy SOD1G93A mice on the congenic C57BL/6 background, treating at 100 mg/kg/day by gavage, starting at 70 days of age. This dose in this specific model has not been assessed previously. Unexpectedly, we report a subset of mice initially administered CuATSM exhibited signs of clinical toxicity, that necessitated euthanasia in extremis after 3-51 days of treatment. Following a 1-week washout period, the remaining mice resumed treatment at the reduced dose of 60 mg/kg/day. At this revised dose, treatment with CuATSM slowed disease progression and increased survival relative to vehicle-treated littermates. This work provides the first evidence that CuATSM produces positive disease-modifying outcomes in high copy SOD1G93A mice on a congenic C57BL/6 background. Furthermore, results from the 100 mg/kg/day phase of the study support dose escalation determination of tolerability as a prudent step when assessing treatments in previously unassessed models or genetic backgrounds.

Selective targeting of 2'-deoxy-5-fluorouridine to urokinase positive malignant cells in vitro.

A urokinase targeting conjugate of 2'-deoxy-5-fluorouridine (5-FUdr) was synthesized and tested for tumor-cell selective cytotoxicity in vitro. The 5-FUdr prodrug 2'-deoxy-5-fluoro-3'-O-(3-carboxypropanoyl)uridine (5-FUdrsuccOH) containing an ester-labile succinate linker was attached to the specific urokinase inhibitor plasminogen activator inhibitor type II (PAI-2) and was found to preferentially kill urokinase-over expressing cancer cells. Up to 7 molecules of 5-FUdr were incorporated per PAI-2 molecule without affecting protein activity. This is the first time a small organic cytotoxin has been conjugated to PAI-2.

N-Alkylisatin-Loaded Liposomes Target the Urokinase Plasminogen Activator System in Breast Cancer.

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.