Micelle-Formulated Juglone Effectively Targets Pancreatic Cancer and Remodels the Tumor Microenvironment.

Pancreatic cancer remains a formidable challenge due to limited treatment options and its aggressive nature. In recent years, the naturally occurring anticancer compound juglone has emerged as a potential therapeutic candidate, showing promising results in inhibiting tumor growth and inducing cancer cell apoptosis. However, concerns over its toxicity have hampered juglone's clinical application. To address this issue, we have explored the use of polymeric micelles as a delivery system for juglone in pancreatic cancer treatment. These micelles, formulated using Poloxamer 407 and D-α-Tocopherol polyethylene glycol 1000 succinate, offer an innovative solution to enhance juglone's therapeutic potential while minimizing toxicity. In-vitro studies have demonstrated that micelle-formulated juglone (JM) effectively decreases proliferation and migration and increases apoptosis in pancreatic cancer cell lines. Importantly, in-vivo, JM exhibited no toxicity, allowing for increased dosing frequency compared to free drug administration. In mice, JM significantly reduced tumor growth in subcutaneous xenograft and orthotopic pancreatic cancer models. Beyond its direct antitumor effects, JM treatment also influenced the tumor microenvironment. In immunocompetent mice, JM increased immune cell infiltration and decreased stromal deposition and activation markers, suggesting an immunomodulatory role. To understand JM's mechanism of action, we conducted RNA sequencing and subsequent differential expression analysis on tumors that were treated with JM. The administration of JM treatment reduced the expression levels of the oncogenic protein MYC, thereby emphasizing its potential as a focused, therapeutic intervention. In conclusion, the polymeric micelles-mediated delivery of juglone holds excellent promise in pancreatic cancer therapy. This approach offers improved drug delivery, reduced toxicity, and enhanced therapeutic efficacy.

Microfluidics Formulated Liposomes of Hypoxia Activated Prodrug for Treatment of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) presents as an unmet clinical challenge for drug delivery due to its unique hypoxic biology. Vinblastine-N-Oxide (CPD100) is a hypoxia-activated prodrug (HAP) that selectively converts to its parent compound, vinblastine, a potent cytotoxic agent, under oxygen gradient. The study evaluates the efficacy of microfluidics formulated liposomal CPD100 (CPD100Li) in PDAC. CPD100Li were formulated with a size of 95 nm and a polydispersity index of 0.2. CPD100Li was stable for a period of 18 months when freeze-dried at a concentration of 3.55 mg/mL. CPD100 and CPD100Li confirmed selective activation at low oxygen levels in pancreatic cancer cell lines. Moreover, in 3D spheroids, CPD100Li displayed higher penetration and disruption compared to CPD100. In patient-derived 3D organoids, CPD100Li exhibited higher cell inhibition in the organoids that displayed higher expression of hypoxia-inducible factor 1 alpha (HIF1A) compared to CPD100. In the orthotopic model, the combination of CPD100Li with gemcitabine (GEM) (standard of care for PDAC) showed higher efficacy than CPD100Li alone for a period of 90 days. In summary, the evaluation of CPD100Li in multiple cellular models provides a strong foundation for its clinical application in PDAC.

A clinically relevant formulation for direct administration of nerve specific fluorophores to mitigate iatrogenic nerve injury.

Iatrogenic nerve injury significantly affects surgical outcomes. Although intraoperative neuromonitoring is utilized, nerve identification remains challenging and the success of nerve sparing is strongly correlated with surgeon experience levels. Fluorescence guided surgery (FGS) offers a potential solution for improved nerve sparing by providing direct visualization of nerve tissue intraoperatively. However, novel probes for FGS face a long regulatory pathway to achieve clinical translation. Herein, we report on the development of a clinically-viable, gel-based formulation that enables direct administration of nerve-specific probes for nerve sparing FGS applications, facilitating clinical translation via the exploratory investigational new drug (eIND) guidance. The developed formulation possesses unique gelling characteristics, allowing it to be easily spread as a liquid followed by rapid gelling for subsequent tissue hold. Optimization of the direct administration protocol with our gel-based formulation enabled a total staining time of 1-2 min for compatibility with surgical procedures and successful clinical translation.

Clinically translatable formulation strategies for systemic administration of nerve-specific probes.

Nerves are extremely difficult to identify and are often accidently damaged during surgery, leaving patients with lasting pain and numbness. Herein, a novel near-infrared (NIR) nerve-specific fluorophore, LGW01-08, was utilized for enhanced nerve identification using fluorescence guided surgery (FGS), formulated using clinical translatable strategies. Formulated LGW01-08 was examined for toxicology, pharmacokinetics (PK), and pharmacodynamics (PD) parameters in preparation for future clinical translation. Optimal LGW01-08 imaging doses were identified in each formulation resulting in a 10x difference between the toxicity to imaging dose window. Laparoscopic swine surgery completed using the da Vinci surgical robot (Intuitive Surgical) demonstrated the efficacy of formulated LGW01-08 for enhanced nerve identification. NIR fluorescence imaging enabled clear identification of nerves buried beneath ~3 mm of tissue that were unidentifiable by white light imaging. These studies provide a strong basis for future clinical translation of NIR nerve-specific fluorophores for utility during FGS to improve patient outcomes.

Hypoxia: Friend or Foe for drug delivery in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal solid tumors with an overall five-year survival rate of that has only just reached 10%. The tumor microenvironment of PDAC is characterized by desmoplasia, which consist of dense stroma of fibroblasts and inflammatory cells, resulting in a hypoxic environment due to limited oxygen diffusion through the tumor. Hypoxia contributes to the aggressive tumor biology by promoting tumor progression, malignancy, and promoting resistance to conventional and targeted therapeutic agents. In depth research in the area has identified that hypoxia modulates the tumor biology through hypoxia inducible factors (HIFs), which not only are the key determinant of pancreatic malignancy but also an important target for therapy. In this review, we summarize the recent advances in understanding hypoxia driven phenotypes, which are responsible for the highly aggressive and metastatic characteristics of pancreatic cancer, and how hypoxia can be exploited as a target for drug delivery.

Select Stabilization of a Tumor-Suppressive PP2A Heterotrimer.

In cancer, suppression of protein phosphatases, such as protein phosphatase 2A (PP2A), that normally counteract kinases, contributes to aberrant signaling. Leonard et al. recently demonstrated that a novel small-molecule activator of PP2A, DT-061, selectively stabilizes a specific PP2A holoenzyme responsible for dephosphorylating critical oncogenic targets, including MYC. The 3.6-Å cryo-electron microscopy map of the heterotrimer assembly provides insight into the druggable structure of PP2A, guiding future phosphatase therapeutics.

Liposomes produced by microfluidics and extrusion: A comparison for scale-up purposes.

Successful liposomal formulations in the clinic are severely limited due to poor translational capability of the traditional bench techniques to clinical production settings. The gold standard for liposome bench manufacturing is a multi-step and parameter dependent extrusion method. Moreover, these parameters need re-optimization for clinical production. The microfluidics technique utilizes vigorous mixing of fluids at a nanoliter scale to produce liposomes in batches from milliliters to a couple liters. The fine control of process parameters results in improved reproducibility between batches. It is inherently scalable; however, the characteristics of liposomes produced by microfluidics both in vitro and in vivo have never been compared to those produced using extrusion. In this manuscript, we describe the comparison between the traditional extrusion method to microfluidics, the new paradigm in liposome production and scale-up.

Liposomal formulation of hypoxia activated prodrug for the treatment of ovarian cancer.

In this work, a new sphingomyelin-cholesterol liposomal formulation (CPD100Li) for the delivery of a hypoxia activated prodrug of vinblastine, mon-N-oxide (CPD100), is developed. The optimized liposomal formulation uses an ionophore (A23187) mediated pH-gradient method. Optimized CPD100Li is characterized for size, drug loading, and stability. The in vitro toxicity of CPD100Li is assessed on different aspects of cell proliferation and apoptosis of ES2 ovarian cancer under normoxic and hypoxic conditions. The pharmacokinetics of CPD100Li in mice as well as the influence of A23187 on the retention of CPD100 are assessed. The dose limiting toxicity (DLT) and maximum tolerated dose (MTD) for CPD100Li are evaluated in nude mice. CPD100 is loaded in the liposome at 5.5 mg/mL. The sizes of CPD100Li using DLS, qNano and cryo-TEM techniques are 155.4 ±â€¯4.2 nm, 132 nm, and 112.6 ±â€¯19.8 nm, respectively. There is no difference between the in vitro characterization of CPD100Li with and without ionophore. Freshly prepared CPD100Li with ionophore are stable for 48 h at 4 °C, while the freeze-dried formulation is stable for 3 months under argon at 4 °C. The hypoxic cytotoxicity ratios (HCR) of CPD100 and CPD100Li are 0.16 and 0.11, respectively. CPD100Li under hypoxic conditions has a 9.2-fold lower IC50 value as compared to CPD100Li under normoxic conditions, confirming the hypoxia dependent activation of CPD100. CPD100Li treated ES2 cells show a time dependent enhanced cell death, along with caspase production and an increase in the number of cells in G0/G1 and higher cell arrest. The blood concentration profile of CPD100Li in mice without A23187 has a 12.6-fold lower area under the curve (AUC) and 1.6-fold lower circulation time compared to the CPD100Li with A23187. The DLT for both CPD100 and CPD100Li is 45 mg/kg and the MTD is 40 mg/kg in nude mice. Based on the preliminary data obtained, we clearly show that the presence of ionophore affects the in vivo stability of CPD100. CPD100Li presents a unique opportunity to develop a first-in-kind chemotherapy product based on achieving selective drug activation through the hypoxic physiologic microenvironment of solid tumors.

Characterization of pegylated and non-pegylated liposomal formulation for the delivery of hypoxia activated vinblastine-N-oxide for the treatment of solid tumors.

Solid tumors often contain hypoxic regions which are resistant to standard chemotherapy and radiotherapy. We have developed a liposomal delivery system for a prodrug of vinblastine (CPD100) which converts to the parent compound only in the presence of lower oxygen levels. As a part of this work we have developed and optimized two formulations of CPD100: one composed of sphingomyelin/cholesterol (55/45; mol/mol) (CPD100Li) and the other composed of sphingomyelin/cholesterol/PEG (55/40/5; mol/mol) (CPD100 PEGLi). We evaluated the antiproliferative effect of CPD100 and the two formulations against A549 non-small lung cancer cell. A549 cell line showed to be sensitive to CPD100 and the two formulations displayed a higher hypoxic: air cytotoxicity ratio compared to the pro-drug. CPD100 elimination from the circulation after injection in mouse was characterized by a very short circulation time (~0.44h), lower area under the curve (AUC) (33µgh/mL) and high clearance (916mL/h/kg) and lower volume of distribution (17.4mL/kg).Total drug elimination from the circulation after the administration of liposomal formulation was characterized by prolonged circulation time (5.5h) along with increase in the AUC (56µgh/mL) for CPD100 Li and (9.5h) with AUC (170µgh/mL) for CPD100PEGLi. This was observed along with increase in volume of distribution and decrease in clearance for the liposomes. The systemic exposure of the free drug was much lower than that achieved with the liposomes. When evaluated for the efficacy in A549 xenograft model in mice, both the liposomes demonstrated excellent tumor suppression and reduction for 3months. The blood chemistry panel and the comprehensive blood analysis showed no increase or decrease in the markers and blood count. In summary, the pharmacokinetic analysis along with the efficacy data emphasis on how the delivery vehicle modifies and enhances the accumulation of the drug and at the same time the increased systemic exposure is not related to toxicity.