Enhancing Oral Delivery of Biologics: A Non-Competitive and Cross-Reactive Anti-Leptin Receptor Nanofitin Demonstrates a Gut-Crossing Capacity in an Ex Vivo Porcine Intestinal Model.

Biotherapeutics exhibit high efficacy in targeted therapy, but their oral delivery is impeded by the harsh conditions of the gastrointestinal (GI) tract and limited intestinal absorption. This article presents a strategy to overcome the challenges of poor intestinal permeability by using a protein shuttle that specifically binds to an intestinal target, the leptin receptor (LepR), and exploiting its capacity to perform a receptor-mediated transport. Our proof-of-concept study focuses on the characterization and transport of robust affinity proteins, known as Nanofitins, across an ex vivo porcine intestinal model. We describe the potential to deliver biologically active molecules across the mucosa by fusing them with the Nanofitin 1-F08 targeting the LepR. This particular Nanofitin was selected for its absence of competition with leptin, its cross-reactivity with LepR from human, mouse, and pig hosts, and its shuttle capability associated with its ability to induce a receptor-mediated transport. This study paves the way for future in vivo demonstration of a safe and efficient oral-to-systemic delivery of targeted therapies.

Targeted Nanofitin-drug Conjugates Achieve Efficient Tumor Delivery and Therapeutic Effect in an EGFRpos Mouse Xenograft Model.

Adjusting the molecular size, the valency and the pharmacokinetics of drug conjugates are as many leverages to improve their therapeutic window, notably by affecting tumor penetration, renal clearance, and short systemic exposure. In that regard, small tumor-targeting ligands are gaining attention. In this study, we demonstrate the benefits of the small Nanofitin alternative scaffolds (7 kDa) as selective tumor-targeting modules for the generation of drug conjugates, focusing on Nanofitins B10 and D8 directed against the EGFR. Owing to their small size and monovalent format, the two Nanofitins displayed a fast and deep tumor penetration in EGFR-positive A431 xenografts in BALB/c nude mice after intravenous administration, yielding to a targeting of respectively 67.9% ± 14.1 and 98.9% ± 0.7 of the tumor cells as demonstrated by IHC. Conjugation with the monomethyl auristatin E toxin provided homogeneous Nanofitin-drug conjugates, with an overall yield of ≥97%, for in vivo assessment in a curative xenograft model using bioluminescent, EGFR-positive, A431 cells in BALB/c nude mice. Internalization was found critical for efficient release of the toxin. Hence, the intravenous administration of the D8-based construct showed significant antitumor effect in vivo as determined by monitoring tumor volumes and bioluminescence levels over 2 months.

Challenges and Opportunities in the Oral Delivery of Recombinant Biologics.

Recombinant biological molecules are at the cutting-edge of biomedical research thanks to the significant progress made in biotechnology and a better understanding of subcellular processes implicated in several diseases. Given their ability to induce a potent response, these molecules are becoming the drugs of choice for multiple pathologies. However, unlike conventional drugs which are mostly ingested, the majority of biologics are currently administered parenterally. Therefore, to improve their limited bioavailability when delivered orally, the scientific community has devoted tremendous efforts to develop accurate cell- and tissue-based models that allow for the determination of their capacity to cross the intestinal mucosa. Furthermore, several promising approaches have been imagined to enhance the intestinal permeability and stability of recombinant biological molecules. This review summarizes the main physiological barriers to the oral delivery of biologics. Several preclinical in vitro and ex vivo models currently used to assess permeability are also presented. Finally, the multiple strategies explored to address the challenges of administering biotherapeutics orally are described.

Platelet Transforming Growth Factor-ß1 Induces Liver Sinusoidal Endothelial Cells to Secrete Interleukin-6.

The roles and interactions of platelets and liver sinusoidal endothelial cells in liver regeneration are unclear, and the trigger that initiates hepatocyte proliferation is unknown. We aimed to identify the key factors released by activated platelets that induce liver sinusoidal endothelial cells to produce interleukin-6 (IL-6), a cytokine implicated in the early phase of liver regeneration. We characterized the releasate of activated platelets inducing the in vitro production of IL-6 by mouse liver sinusoidal endothelial cells and observed that the stimulating factor was a thermolabile protein. Following gel filtration, a single fraction of activated platelet releasate induced a maximal IL-6 secretion by liver sinusoidal endothelial cells (90.2 ± 13.9 versus control with buffer, 9.0 ± 0.8 pg/mL, p < 0.05). Mass spectroscopy analysis of this fraction, followed by in silico processing, resulted in a reduced list of 18 candidates. Several proteins from the list were tested, and only recombinant transforming growth factor ß1 (TGF-ß1) resulted in an increased IL-6 production up to 242.7 ± 30.5 pg/mL, which was comparable to non-fractionated platelet releasate effect. Using neutralizing anti-TGF-ß1 antibody or a TGF-ß1 receptor inhibitor, IL-6 production by liver sinusoidal endothelial cells was dramatically reduced. These results support a role of platelet TGF-ß1 ß1 in the priming phase of liver regeneration.

The autophagy inhibitor verteporfin moderately enhances the antitumor activity of gemcitabine in a pancreatic ductal adenocarcinoma model.

Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to chemotherapy. It has been described as requiring elevated autophagy for growth and inhibiting autophagy has been proposed as a treatment strategy. To date, all preclinical reports and clinical trials investigating pharmacological inhibition of autophagy have used chloroquine or hydroxychloroquine, which interfere with lysosomal function and block autophagy at a late stage. Verteporfin is a newly discovered autophagy inhibitor that blocks autophagy at an early stage by inhibiting autophagosome formation. Here we report that PDAC cell lines show variable sensitivity to verteporfin in vitro, suggesting cell-line specific autophagy dependence. Using image-based and molecular analyses, we show that verteporfin inhibits autophagy stimulated by gemcitabine, the current standard treatment for PDAC. Pharmacokinetic and efficacy studies in a BxPC-3 xenograft mouse model demonstrated that verteporfin accumulated in tumors at autophagy-inhibiting levels and inhibited autophagy in vivo, but did not reduce tumor volume or increase survival as a single agent. In combination with gemcitabine verteporfin moderately reduced tumor growth and enhanced survival compared to gemcitabine alone. While our results do not uphold the premise that autophagy inhibition might be widely effective against PDAC as a single-modality treatment, they do support autophagy inhibition as an approach to sensitize PDAC to gemcitabine.

In vitro and in vivo evaluation of intravesical docetaxel loaded hydrophobically derivatized hyperbranched polyglycerols in an orthotopic model of bladder cancer.

The objective of this study was to evaluate the tolerability, to establish a dosing regimen, and to evaluate the efficacy of intravesical docetaxel (DTX) formulations in a mouse model of bladder cancer. DTX in commercial formulation (Taxotere, DTX in Tween 80) or loaded in hyperbranched polyglycerols (HPGs) was evaluated. The synthesis and characterization of HPGs with hydrophobic cores and derivatized with methoxy poly(ethylene glycol) in the shell and further functionalized with amine groups (HPG-C(8/10)-MePEG and HPG-C(8/10)-MePEG-NH(2)) is described. Intravesical DTX in either commercial or HPGs formulations (up to 1.0 mg/mL) was instilled in mice with orthotopic bladder cancer xenografts and was well tolerated with no apparent signs of local or systemic toxicities. Furthermore, a single dose of intravesical DTX (0.5 mg/mL) loaded in HPGs was significantly more effective in reducing the tumor growth in an orthotopic model of bladder cancer than the commercial formulation of Taxotere. In addition, DTX-loaded HPG-C(8/10)-MePEG-NH(2) was found to be more effective at lower instillation dose than DTX (0.2 mg/mL)-loaded HPG-C(8/10)-MePEG. Overall, our data show promising antitumor efficacy and safety in a recently validated orthotopic model of bladder cancer. Further research is warranted to evaluate its safety and efficacy in early phase clinical trials in patients refractory to standard intravesical therapy.

MPEG-hexPLA micelles as novel carriers for hypericin, a fluorescent marker for use in cancer diagnostics.

Ovarian cancer is the most common gynecological cancer diagnosed in Western countries. Detection of micrometastases at an early stage of the disease could lead to a cure rate of 90% by limiting the spread of the disease outside the ovaries. In this article, hypericin (Hy), a hydrophobic photosensitizer used for the photodynamic diagnosis (PD) of ovarian cancer, was efficiently incorporated into a core of micelles made from methoxy-poly(ethylene glycol) and hexyl-substituted poly(lactides) copolymers. The fate of these micelles following intravenous injection was studied in vivo in two ovarian tumor-bearing animal models. In the chick embryo chorioallantoic membrane model, 17 times more Hy accumulated in tumor nodules when Hy was delivered with micelles than when Hy was delivered as an ethanol solution. Studies of the biodistribution of Hy in Fisher rats revealed escape of these nanosized micelles (<32 nm) from the mononuclear phagocyte system. Hy-loaded micelles showed maximal accumulation in tumors and demonstrated the best tumor/muscle contrast visible 3 h after injection in the rat model. The rapid and highly selective accumulation of Hy in tumors that we demonstrated in this study suggests that these micelle formulations could be used for the PD of ovarian cancer in the future.

Quenching-induced deactivation of photosensitizer by nanoencapsulation to improve phototherapy of cancer.

Photodynamic therapy has emerged as a promising alternative to current cancer treatment. However, conventional photosensitizers have several limitations due to their unsuitable pharmaceutical formulations and lack of selectivity. Our strategy was to exploit the advantages of nanoparticles and the quenching-induced deactivation of the model photosensitizer hypericin to produce "activatable" drug delivery systems. Efficient fluorescence and activity quenching were achieved by increasing the drug-loading rate of nanoparticles. In vitro assays confirmed the reversibility of hypericin deactivation, as the hypericin fluorescence and photodynamic activity were recovered upon cell internalization.

Screening of nanoparticulate delivery systems for the photodetection of cancer in a simple and cost-effective model.

AIMS: In urology, fluorescence-based imaging methods have been proven to significantly improve the detection of small, barely visible tumors and reduce the recurrence rate. Under ethical and economical pressure, new effective screening systems have to be developed to exploit and assess novel strategies for fluorescence photodetection in other areas. For this purpose, the chorioallantoic membrane (CAM) of the developing chick embryo is an attractive alternative model to the mammalian models. MATERIALS & METHODS: Hypericin encapsulated into nanoparticles for the photodetection of ovarian metastases was evaluated in the CAM model with respect to vascular extravazation and tumor targeting and compared with free drug following intravenous administration. RESULTS: To validate the CAM model as a valuable screening system for photodetection of cancer, we drew a comparison with results obtained on a conventional rodent model. CONCLUSION: Rodent and CAM models led to the same conclusion regarding the benefits of nanoencapsulation to improve selective accumulation of drug in ovarian micrometastases.

Benefits of nanoencapsulation for the hypercin-mediated photodetection of ovarian micrometastases.

The high recurrence and lethality of ovarian cancer at advanced stages is problematic, especially due to the development of numerous micrometastases scattered throughout the abdominal cavity. Fluorescence photodetection (PD) used in combination with surgical resection of malignant tissues has been suggested to improve recovery. Based on promising in vivo results for the detection of bladder cancer, hypericin (Hy), a natural photosensitizer (PS), stands as a good candidate for the photodetection of ovarian cancer. However, due to its hydrophobicity, systemic administration of Hy is problematic. Polymeric nanoparticles (NPs) help to overcome these delivery and stability problems and enable intravenous administration of Hy. In this study, Hy-loaded NPs of polylactic acid were produced with the following properties: (i) mean size of 268 nm, (ii) negative zeta potential, (iii) low residual surfactant and (iv) drug loading of 3.7 % (w/w). The potential of hypericin-loaded nanoparticles for the fluorescence photodetection of ovarian metastases in Fischer 344 rats bearing ovarian tumours was compared to free drug. The selectivity of Hy administered with both formulations was assessed first by fluorescence endoscopy, and then quantified after tissue extraction. The results showed an improved selective accumulation of Hy in ovarian micrometastases when NPs were used.

The chick embryo and its chorioallantoic membrane (CAM) for the in vivo evaluation of drug delivery systems.

Mammalian models are frequently used for preclinical evaluation of new drug delivery systems (DDS). However, valid mammalian models are expensive, time-consuming, and not easy to set up and evaluate. Furthermore, they are often linked to administrative burden with respect to ethical and legal aspects. The present review outlines the possibilities and limitations of using the hen's embryo, and specifically its chorioallantoic membrane (CAM), as an alternative to mammalian models for the evaluation of DDS. Features of the CAM, the anatomy of the embryo, and the blood were investigated to assess properties of the drug carriers such as toxicity and biocompatibility, as well as the activity, toxicity, biodistribution and pharmacokinetics of the drug. The simplicity, rapidity, and low cost of the different assays that can be performed with chick embryos strengthen the interest of routinely using this model in pharmaceutical technology research. It is concluded that there is a big potential for using chick embryos in screening procedures of formulation candidates, thus establishing an intermediate step between in vitro cellular tests and preclinical mammalian models.

Hypericin-loaded nanoparticles for the photodynamic treatment of ovarian cancer.

A photodynamic approach has been suggested to improve diagnosis and therapy of ovarian cancer. As Hypericin (Hy), a natural photosensitizer (PS) extracted from Hypericum perforatum, has been shown to be efficient in vitro and in vivo for the detection or treatment of other cancers, Hy could also be a potent tool for the treatment and detection of ovarian cancer. Due to its hydrophobicity, systemic administration of Hy is problematic. Thus, polymeric nanoparticles (NPs) of polylactic acid (PLA) or polylactic-co-glycolic acid (PLGA) were used as a drug delivery system. Hy-loaded NPs were produced with the following characteristics: (i) size in the 200-300 nm range, (ii) negative zeta potential, (iii) low residual PVAL and (iv) drug loading from 0.03 to 0.15% (w/w). Their in vitro photoactivity was investigated on the NuTu-19 ovarian cancer cell model derived from Fischer 344 rats and compared to free drug. Hy-loaded PLA NPs exhibited a higher photoactivity than free drug. Increasing light dose or incubation time with cells induced an enhanced activity of Hy-loaded PLA NPs. Increased NP drug loading had a negative effect on their photoactivity on NuTu-19 cells: at the same Hy concentration, the higher was the drug loading, the lower was the phototoxic effect. The influence of NP drug loading on the Hy release from NPs was also investigated.