Folate receptor targeted nanoparticles containing niraparib and doxorubicin as a potential candidate for the treatment of high grade serous ovarian cancer.

Combination chemotherapy is an established approach used to manage toxicities while eliciting an enhanced therapeutic response. Delivery of drug combinations at specific molar ratios has been considered a means to achieve synergistic effects resulting in improvements in efficacy while minimizing dose related adverse drug reactions. The benefits of this approach have been realized with the FDA approval of Vyxeos®, the first liposome formulation to deliver a synergistic drug combination leading to improved overall survival against standard of care. In the current study, we demonstrate the synergistic potential of the PARP inhibitor niraparib and doxorubicin for the treatment of ovarian cancer. Through in vitro screening in a panel of ovarian cancer cell lines, we find that niraparib and doxorubicin demonstrate consistent synergy/additivity at the majority of evaluated molar ratio combinations. Further to these findings, we report formulation of a nanoparticle encapsulating our identified synergistic combination. We describe a rational design process to achieve highly stable liposomes that are targeted with folate to folate-receptor-alpha, which is known to be overexpressed on the surface of ovarian cancer cells. With this approach, we aim to achieve targeted delivery of niraparib and doxorubicin at a pre-determined synergistic molar ratio via increased receptor-mediated endocytosis.

Triggered release from thermosensitive liposomes improves tumor targeting of vinorelbine.

Triggered drug delivery strategies have been shown to enhance drug accumulation at target diseased sites in comparison to administration of free drug. In particular, many studies have demonstrated improved targetability of chemotherapeutics when delivered via thermosensitive liposomes. However, most studies continue to focus on encapsulating doxorubicin while many other drugs would benefit from this targeted and localized delivery approach. The proposed study explores the therapeutic potential of a thermosensitive liposome formulation of the commonly used chemotherapy drug vinorelbine in combination with mild hyperthermia (39-43 °C) in a murine model of rhabdomyosarcoma. Rhabdomyosarcoma, the most common soft tissue sarcoma in children, is largely treated using conventional chemotherapy which is associated with significant adverse long-term sequelae. In this study, mild hyperthermia was pursued as a non-invasive, non-toxic means to improve the efficacy and safety profiles of vinorelbine. Thorough assessment of the pharmacokinetics, biodistribution, efficacy and toxicity of vinorelbine administered in the thermosensitive liposome formulation was compared to administration in a traditional, non-thermosensitive liposome formulation. This study shows the potential of an advanced formulation technology in combination with mild hyperthermia as a means to target an untargeted therapeutic agent and result in a significant improvement in its therapeutic index.

Shifting the Paradigm on Cannabis Safety.

The global movement toward legalization of cannabis is resulting in an ever-increasing public perception that cannabis is safe. Cannabis is not the first drug to be available for nonmedical use, nor is it the first to have such an unfounded safety profile. The safety of long-term exposure to phytocannabinoids is misunderstood by, and under reported to, the general public. There is evidence to suggest that long-term use of recreational cannabis may be associated with an increased risk of undesirable side effects. This evidence warrants both appropriate caution from the general public and investment in further research by government and industry sectors that are profiting from the sale of these potent psychoactive agents. There is no doubt that these compounds have medical potential. However, in addition to the medical potential, we must also remain aware of the adverse health effects that are becoming synonymous with recreational cannabis use. This perspective highlights the privileged role that cannabis has as a perceived "safe drug" in society and summarizes some concerning side effects that are becoming associated with regular nonprescribed cannabis use.

Turning down the heat: The case for mild hyperthermia and thermosensitive liposomes.

"A single disappointing study does not mean an end to the future of ThermoDox®", writes Michael Tardugno (CEO of Celsion Corporation), after announcing the termination of Celsion's second Phase III clinical trial. The OPTIMA trial, as it was known, evaluated their thermosensitive liposome (TSL) formulation of doxorubicin (ThermoDox®) in combination with radiofrequency ablation for the treatment of hepatocellular carcinoma (HCC). The purpose of this perspective is to review the case of ThermoDox and to address questions related to its clinical translation. Specifically, what has prevented the clinical translation of this once highly regarded breakthrough technology? Is this the end of TSLs? What can we learn from the challenges faced in the clinical development of this multi-modal therapy? As formulation scientists working in the field, we continue to believe that heat-triggered drug delivery platforms have tremendous potential as chemotherapy. Herein, we highlight potential limitations in the design of many of the Thermodox clinical trials, and we propose that despite these setbacks, TSLs have the potential to become an effective component of cancer therapy.

Development and pharmacokinetic evaluation of a self-nanoemulsifying drug delivery system for the oral delivery of cannabidiol.

The number of lipophilic drug candidates in pharmaceutical discovery pipelines has increased in recent years. These drugs often possess physicochemical properties that result in poor oral bioavailability, and their clinical potential may be limited without adequate formulation strategies. Cannabidiol (CBD) is an excellent example of a highly lipophilic compound with poor oral bioavailability, due to low water solubility and extensive first-pass metabolism. An approach that may overcome these limitations is formulation of the drug in self-nanoemulsifying drug delivery systems (SNEDDS). Herein, CBD-SNEDDS formulations were prepared and evaluated in vitro. Promising formulations (F2, F4) were administered to healthy female Sprague-Dawley rats via oral gavage (20 mg/kg CBD). Resulting pharmacokinetic parameters of CBD were compared to those obtained following administration of CBD in two oil-based formulations: a medium-chain triglyceride oil vehicle (MCT-CBD), and a sesame oil-based formulation similar in composition to an FDA-approved formulation of CBD, Epidiolex® (SO-CBD). Compared to MCT-CBD, administration of the SNEDDS formulations led to more rapid absorption of CBD (median Tmax values: 0.5 h (F2), 1 h (F4), 6 h (MCT-CBD)). Administration of F2 and F4 formulations also improved the systemic exposure to CBD by 2.2 and 2.8-fold compared to MCT-CBD; however, no improvement was found compared to SO-CBD.

Heat-activated nanomedicine formulation improves the anticancer potential of the HSP90 inhibitor luminespib in vitro.

The heat shock protein 90 inhibitor, luminespib, has demonstrated potent preclinical activity against numerous cancers. However, clinical translation has been impeded by dose-limiting toxicities that have necessitated dosing schedules which have reduced therapeutic efficacy. As such, luminespib is a prime candidate for reformulation using advanced drug delivery strategies that improve tumor delivery efficiency and limit off-target side effects. Specifically, thermosensitive liposomes are proposed as a drug delivery strategy capable of delivering high concentrations of drug to the tumor in combination with other chemotherapeutic molecules. Indeed, this work establishes that luminespib exhibits synergistic activity in lung cancer in combination with standard of care drugs such as cisplatin and vinorelbine. While our research team has previously developed thermosensitive liposomes containing cisplatin or vinorelbine, this work presents the first liposomal formulation of luminespib. The physico-chemical properties and heat-triggered release of the formulation were characterized. Cytotoxicity assays were used to determine the optimal drug ratios for treatment of luminespib in combination with cisplatin or vinorelbine in non-small cell lung cancer cells. The formulation and drug combination work presented in this paper offer the potential for resuscitation of the clinical prospects of a promising anticancer agent.

Poly(δ-valerolactone-co-allyl-δ-valerolactone) cross-linked microparticles: Formulation, characterization and biocompatibility.

A novel polymeric material, poly(δ-valerolactone-co-allyl-δ-valerolactone) (PVL-co-PAVL), was used to manufacture microparticles (MPs) for sustained drug delivery. PVL-co-PAVL MPs were formulated using a modified oil-in-water approach, followed by a UV-initiated cross-linking process. Prepared MPs had a smooth spherical morphology and cross-linking of the copolymer was found to improve the integrity and thermal stability of the MPs. Paclitaxel (PTX) was successfully loaded into the MPs at a high drug loading capacity, using a post-loading swelling-equilibrium method. In vitro evaluation showed that the PVL-co-PAVL MPs provide sustained release of PTX, which exhibited first-order release kinetics. A subsequent pilot pharmacokinetic study was carried out on the PTX-loaded PVL-co-PAVL MPs. During this study, serum levels of PTX were monitored following subcutaneous administration of the MPs to Sprague-Dawley rats. Overall, the in vivo release of PTX from the MPs was lower than expected based on the in vitro release studies. Detectable serum levels of PTX suggest that sustained release of drug was achieved in vivo. Minimal changes in subcutaneous tissue were observed at the site of injection. Future studies will further examine the localized and systemic distribution of drug following administration in this new polymer-based MP system.

Cross-linked valerolactone copolymer implants with tailorable biodegradation, loading and in vitro release of paclitaxel.

Implantable drug delivery systems, formed from degradable and non-degradable polymers, can offer several advantages over traditional dosage forms for sustained drug delivery. The majority of degradable implant systems developed to date are composed of poly(lactide-co-glycolide) (PLGA). However, PLGA-based systems are not suitable for the delivery of all drugs. Each drug is unique in terms of physico-chemical properties, and polymer-drug compatibility plays a significant role in determining a drug formulation's performance. In this study, two novel cross-linkable δ-valerolactone-based copolymers were synthesized and used to prepare cross-linked disc-shaped implants. The manipulation of the composition of the discs and conditions used during drug loading were found to influence various aspects of the delivery system performance including the degree of swelling, degradation, drug-loading and in vitro release. The polymeric discs resulted in no adverse effects following subcutaneous implantation in naïve rats. These studies support further development of cross-linkable valerolactone matrices as implantable formulations for sustained drug delivery.

Dual-Targeted Delivery of Nanoparticles Encapsulating Paclitaxel and Everolimus: a Novel Strategy to Overcome Breast Cancer Receptor Heterogeneity.

PURPOSE: The intratumoral heterogeneity observed in breast cancer (BC), in particular with regard to cell surface receptor expression, can hinder the success of many targeted cancer therapies. The development of novel therapeutic agents that target more than one receptor can overcome this inherent property of tumors and can facilitate their selective internalization in cancer cells. The goal of this study is to develop a drug combination-loaded nanoparticle (NP) formulation that is actively-targeted to HER2 and EGFR receptors on BC cells. METHODS: A polymeric NP formulation was prepared which co-encapsulated a synergistic combination of the chemotherapeutic agent, paclitaxel (PTX), and the mTOR inhibitor, everolimus (EVER), and is targeted to HER2 and EGFR receptors on BC cells using antibody Fab fragments as the targeting moieties. The physicochemical characteristics of the dual-targeted formulation (Dual-NP) were evaluated, along with its cytotoxic profile (in both, monolayer and 3D BC models), as well as the degree of cellular uptake in HER2high/EGFRmod and HER2neg/EGFRlow BC cells. RESULTS: Dual-NPs were found to have significantly higher cytotoxicity relative to HER2 mono-targeted (T-NPs) and untargeted NPs (UT-NPs) in HER2high/EGFRmod monolayer BC cells after 72 h exposure, while no significant difference was observed in HER2neg/EGFRlow cells. However, in the HER2high/EGFRmod spheroids, the cytotoxicity of Dual-NPs was comparable to that of T-NPs. This was thought to be attributed to the previously reported downregulation of EGFR in 3D in comparison to 2D BC models. Dual-NPs had significantly higher cellular uptake relative to UT-NPs and T-NPs in HER2high/EGFRmod BC cells after 24 h exposure, whereas in the HER2neg/EGFRlow cells, the increase in cellular uptake of the Dual-NPs was not as high as the level achieved in the HER2high/EGFRmod cells. Blocking HER2 and EGFR significantly reduced the uptake of T-NPs and Dual-NPs in the HER2high/EGFRmod BC cells, demonstrating specific binding to both EGFR and HER2. CONCLUSIONS: The dual-targeting strategy developed in this study in conjunction with a potentially promising delivery vector for a synergistic combination therapy can overcome receptor heterogeneity, yielding significant improvements in the cytotoxicity and cellular uptake in BC cells.

Development of a Bioluminescent BRCA1-Deficient Xenograft Model of Disseminated, High-Grade Serous Ovarian Cancer.

Successful translation of preclinical data relies on valid and comprehensive animal models. While high-grade serous ovarian cancer (HGSOC) is the most prevalent subtype, the most commonly used ovarian cancer cell lines are not representative of HGSOC. In addition, 50% of ovarian cancer patients present with dysfunctional BRCA1/2, however currently there is a shortage of BRCA-deficient models. By utilizing the OVCAR8 cell line, which contains a hypermethylated BRCA1 promoter, the aim of the current study was to establish and characterize an animal model for BRCA-deficient HGSOC. Transfection of the luciferase gene to OVCAR8 cells enabled bioluminescent imaging for real-time, non-invasive monitoring of tumor growth. The resulting model was characterized by peritoneal metastasis and ascites formation at late stages of disease. Immunohistochemical staining revealed high-grade serous histology in all resected tumor nodules. Immunoblotting and qPCR analysis demonstrated BRCA1 deficiency was maintained in vivo. Moderate to strong correlations were observed between bioluminescent signal and tumor weight. Lastly, intraperitoneal administration of carboplatin significantly reduced tumor growth as measured by bioluminescence. The current model demonstrated BRCA1 deficiency and a high resemblance of the clinical features of HGSOC. This model may be well-suited for evaluation of therapeutic efficacy in BRCA-deficient HGSOC.

Nutritional and Post-Transplantation Outcomes of Enteral versus Parenteral Nutrition in Pediatric Hematopoietic Stem Cell Transplantation: A Systematic Review of Randomized and Nonrandomized Studies.

Hematopoietic stem cell transplantation (HSCT) involves the administration of chemotherapy followed by the infusion of donor stem cells. After treatment, children can consequently experience nausea, vomiting, diarrhea, anorexia, and mucositis, which negatively impact oral intake, leading to rapid deterioration in nutritional status and risk of malnutrition. Nutrition support therefore becomes necessary to circumvent these adverse effects. This has traditionally been provided via parenteral nutrition (PN), but pediatric evidence is increasingly advocating enteral nutrition (EN) as a preferential alternative. The objective of this review is to determine the efficacy of any forms of EN versus PN provided during admission to children aged ≤ 18 years undergoing HSCT. Primary outcomes considered efficacy in relation to various nutritional parameters, and secondary outcomes included a range of post-transplantation parameters. Data sources included English and non-English articles from the start date of MEDLINE, EMBASE, AMED, CINAHL and Cochrane Controlled Trials register, up to July 2018. Key journals were also hand searched, reference lists scanned, clinical experts contacted, and gray literature searched using EThOS and Open Grey. Randomized and observational studies comparing any forms of EN versus PN in children aged ≤ 18 years undergoing HSCT investigating nutritional or post-transplantation outcomes were eligible. Data were extracted from included studies using a custom extraction form that had previously been piloted. Because included studies were observational, risk of bias was assessed using Risk of Bias in Non-randomised Studies of Interventions. Because only a small number of heterogenous studies reporting a wide range of differently defined outcomes were included, meta-analyses were not performed and data were presented in narrative form. Conflicting results in favor of either method of nutrition support or no difference between methods were seen for duration of interventions, nutritional intakes, biochemical and anthropometric changes, mortality, infections, length of admission, and neutrophil engraftment. EN may provide favorable benefits over PN regarding acute graft-versus-host-disease (aGVHD) and platelet engraftment. A paucity of studies was found investigating the question posed by this review. Included studies were clinically heterogenous regarding populations, interventions, and outcomes, at moderate to serious risk of bias due to the absence of randomization, confounding parameters, statistical control, retrospective designs, and participant selection. Some studies were more than 15 years old. Despite the limited number and poor quality of identified studies, results support the growing body of pediatric evidence that EN is feasible during HSCT. Similar differences regarding many nutritional and post-transplantation outcomes were seen in both forms of nutrition support, but EN could provide benefits above PN including reduced incidence of aGVHD and faster platelet engraftment.

Lipids and polymers in pharmaceutical technology: Lifelong companions.

In pharmaceutical technology, lipids and polymers are considered pillar excipients for the fabrication of most dosage forms, irrespective of the administration route. They play various roles ranging from support vehicles to release rate modifiers, stabilizers, solubilizers, permeation enhancers and transfection agents. Focusing on selected applications, which were discussed at the Annual Scientific Meeting of the Gattefossé Foundation 2018, this manuscript recapitulates the fundamental roles of these two important classes of excipients, either employed alone or in combination, and provides insight on their functional properties in various types of drug formulations. Emphasis is placed on oral formulations for the administration of active pharmaceutical ingredients with low aqueous solubilities or poor permeation properties. Additionally, this review article covers the use of lipids and polymers in the design of colloidal injectable delivery systems, and as substrates in additive manufacturing technologies for the production of tailor-made dosage forms.

Codelivery of Paclitaxel and Everolimus at the Optimal Synergistic Ratio: A Promising Solution for the Treatment of Breast Cancer.

Clinical studies examining the combination of paclitaxel (PTX) and everolimus (EVER), an mTOR inhibitor, have failed to result in significant improvements in efficacy and toxicity in patients with breast cancer (BC), relative to treatment with PTX alone. These disappointing clinical trial results have been attributed to poorly designed preclinical studies using the combination of PTX and EVER as well as the significantly different pharmacokinetic profiles of the two drugs. In the current work, the potential synergy between PTX and EVER was examined in a panel of six BC cell lines that differ in terms of their molecular subtype and drug sensitivity. Polymeric nanoparticles (NPs) were used to encapsulate PTX and EVER at an optimal synergistic ratio to achieve specific, colocalized delivery of the combination therapy in BC cell lines. Combinations of PTX and EVER (especially at relatively high doses of EVER) resulted in pronounced synergy in all BC cell lines evaluated. The optimal molar ratio of PTX:EVER was determined to be 1:0.5. The combination was delivered to BC cells at the synergistic ratio via encapsulation within polymeric NPs formed from the poly(ethylene glycol)- b-poly(lactide- co-glycolide) (PEG- b-PLGA) copolymer. The NPs had an average diameter of less than 100 nm and were capable of in vitro retention of the encapsulated PTX and EVER at the optimal synergistic molar ratio for over 7 days. Cytotoxicity data demonstrated that PTX+EVER-loaded NPs were significantly less cytotoxic than the free drug combination in MCF-7 and SKBR3 BC cell lines following 72 h, suggesting that PTX+EVER-loaded NPs remain stable and retain the drug combination loaded within the core after 72 h. The uptake of FITC-labeled NPs in SKBR3 cells was evaluated by flow cytometry, with approximately 41% of cells demonstrating detectable fluorescence after 24 h of exposure. The thorough and systematic approach used in this study to determine and evaluate a synergistic PTX:EVER ratio in conjunction with a potentially promising delivery vector for the drug combination could offer a future clinical benefit for patients with BC.

BRCA Status Does Not Predict Synergism of a Carboplatin and Olaparib Combination in High-Grade Serous Ovarian Cancer Cell Lines.

Over 50% of epithelial ovarian cancers express the BRCAness profile that leads to a dysfunctional homologous recombination repair system. The combination of a dysfunctional homologous recombination repair system and a poly(ADP-ribose) polymerase (PARP) inhibitor results in a synthetic lethal phenotype. The PARP inhibitor olaparib, approved as a monotherapy for patients with a germline BRCA mutation, has shown promising results in preclinical studies when combined with DNA damaging agents, such as carboplatin. However, dose-limiting toxicities have hindered the use of a combination therapy with olaparib in the clinical setting. By concurrent administration of carboplatin and olaparib at various molar ratios of drugs, the aim of this study was to explore the optimal dosing ratio of carboplatin-olaparib combinations in a comprehensive panel of eight BRCA-proficient and -deficient high-grade serous ovarian cancer (HGSOC) cell lines. Overall, synergy was observed in the BRCA1/2-mutated or defective cell lines when olaparib was combined at lower molar ratios of olaparib to carboplatin. Immunostaining of γH2AX foci revealed increased DNA damage as a result of this synergistic drug combination in the UWB1.289 paired cell lines. In vitro activity of the individual agents, carboplatin and olaparib, did not correlate with PARP1 expression in each cell line. Importantly, synergism was also observed in a subset of BRCA wild-type cell lines (OV90 and PEO4) suggesting therapeutic benefits of this combination beyond BRCA-dependent synthetic lethality. The administration of drugs at synergistic ratios has the potential to increase efficacy and reduce toxicity.

Cyclodextrin-siRNA conjugates as versatile gene silencing agents.

Functional siRNAs (luciferase and PLK1) have been conjugated to ß-cyclodextrin and the ability of the conjugates to retain gene knockdown activity has been assessed by delivery to cancer cell lines using various formulations. Initially two formulations used complexation with polycations, namely Lipofectamine 2000 and an amphiphilic polycationic cyclodextrin. Gene knockdown results for human glioblastoma cells (U87) and prostate cancer cells (PC3, DU145) showed that conjugation to the cyclodextrin did not reduce gene silencing by the RNA. A third mode of delivery involved formation of targeted nanoparticles in which the conjugate was first complexed with adamantyl-PEG-ligands (targeting ligand RVG peptide or dianisamide) by adamantyl inclusion in the cyclodextrin cavities of the conjugates, followed by charge neutralisation with the cationic polymer chitosan. Enhanced knockdown was achieved by these ligand-targeted formulations. In summary, while this study illustrated the gene silencing efficacy of a simple cyclodextrin-siRNA conjugate it is envisaged that future studies will explore the use of conjugates with a modified cyclodextrin which would be self-delivering. Detailed data such as stability, lysosomal escape etc. will then be reported for each conjugate, since this will be appropriate for conjugates which are intended to exploit, rather than merely demonstrate, the concept. The present paper was intended to demonstrate the viability and generality of this novel concept.

Ratio-Dependent Synergism of a Doxorubicin and Olaparib Combination in 2D and Spheroid Models of Ovarian Cancer.

Ovarian cancer is the fourth leading cause of death in women in developed countries. Even though patients with the most lethal form of the disease (HGSOC; high grade serous ovarian cancer) respond well to initial treatment, they often relapse with progressively resistant disease. Inhibitors of the poly(ADP-ribose) polymerase (PARP) enzymes are a relatively new class of molecularly targeted small molecule drugs that show promise in overcoming resistance. The present study explores the combination of a DNA damaging agent, doxorubicin (DOX), with the PARP inhibitor, olaparib (OLP), in order to achieve optimal synergy of both drugs in serous ovarian cancer. This drug combination was evaluated and optimized in 2D monolayers and 3D multicellular tumor spheroids (MCTS) using a genetically and histologically characterized panel of nine OC cell lines with or without BRCA1 or BRCA2 mutations. Combination index (CI) values of DOX and OLP were determined using the Chou and Talalay method. The potency of this drug combination was found to rely heavily on the molar ratios at which the two drugs are combined. In general, MCTS growth inhibition was reflective of the patterns predicted by the CI values obtained in monolayers. Promising combination ratios identified in this study warrant further preclinical and clinical investigation.

Folate-targeted amphiphilic cyclodextrin nanoparticles incorporating a fusogenic peptide deliver therapeutic siRNA and inhibit the invasive capacity of 3D prostate cancer tumours.

The main barrier to the development of an effective RNA interference (RNAi) therapy is the lack of a suitable delivery vector. Modified cyclodextrins have emerged in recent years for the delivery of siRNA. In the present study, a folate-targeted amphiphilic cyclodextrin was formulated using DSPE-PEG5000-folate to target prostate cancer cells. The fusogenic peptide GALA was included in the formulation to aid in the endosomal release of siRNA. Targeted nanoparticles were less than 200nm in size with a neutral surface charge. The complexes were able to bind siRNA and protect it from serum nucleases. Incubation with excess free folate resulted in a significant decrease in the uptake of targeted nanoparticles in LNCaP and PC3 cells, both of which have been reported to have differing pathways of folate uptake. There was a significant reduction in the therapeutic targets, ZEB1 and NRP1 at mRNA and protein level following treatment with targeted complexes. In preliminary functional assays using 3D spheroids, treatment of PC3 tumours with targeted complexes with ZEB1 and NRP1 siRNA resulted in more compact colonies relative to the untargeted controls and inhibited infiltration into the Matrigel™ layer.