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Exosomes are biological nano-sized vesicles (~30-200 nm in diameter) that are produced by a wide range of cells and play several roles in cell-cell communications. These vesicles contain membrane and cytoplasmic components of producing cells. Mesenchymal stem cells (MSCs) are the ideal producer of exosomes. The secreted vesicles from MSCs are promising biological vehicles for cell-free therapy in regenerative medicine, cancer therapy and targeted delivery of therapeutic agents to the tumor cells. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising member of the TNF family with selective effect on cancerous cells. Recent evidences showed that the membrane TRAIL-armed exosomes possess anti-tumor activity. However, the effect of in vivo administration of TRAIL-armed exosomes has not been reported so far. In the current study, mesenchymal stem cells expressing TRAIL/GFP proteins were prepared with the help of a non-viral vector based on polyethylenimine 25 kDa. Then, exosomes containing TRAIL protein (Exo-TRAIL) were isolated from the supernatant of genetically engineered MSCs and characterized. Antitumor activity of both MSC-derived exosomes and Exo-TRAIL was investigated in vitro and in vivo in three models. The results indicated that the co-injection of both Exo-TRAIL and tumor cells delayed the tumor appearance. Besides, the tumor volume/weight was efficiently decreased in tumor bearing mice. Moreover, it was shown that multi-dose injections of Exo-TRAIL reduced the tumor size while single dose treatment with Exo-TRAIL did not show significant anti-tumor activity. To conclude, these results suggested that MSC-derived Exo-TRAIL has a potential capacity for cancer treatment. [corrected].

Objectives: Circularly permuted tumor necrosis factor-related apoptosis-inducing ligand (CPT), or CPT, is a novel antitumor drug candidate. This phase 1b study evaluated the safety, tolerability, pharmacokinetics (PK), and efficacy of single-agent CPT in patients with relapsed or refractory multiple myeloma (RRMM), and aimed to identify the recommended dose for the phase 2 study., Materials and Methods: Patients received single or multiple doses (once daily for 5 consecutive days per 21-d cycle) of CPT intravenous infusion at doses of 5, 6.5, 8, 10, and 15 mg/kg, to determine the maximum tolerated dose, dose-limiting toxicities, safety, and tolerability. PK were evaluated. Preliminary efficacy was assessed after each treatment cycle., Results: Twenty-nine RRMM patients received CPT. Neither the dose-limiting toxicity nor the maximum tolerated dose were identified. The most common treatment-related adverse events were liver enzyme elevations (eg, elevation of aspartate aminotransferase and alanine aminotransferase), hematological abnormalities (eg, leukopenia and neutropenia), fever, fatigue, and vomiting. CPT had a terminal half-life of 0.90 to 1.27 hours at the 5 dose levels, and no accumulation was observed with repeated doses. Safety and PK profiles were similar across the 5 dose cohorts. The overall response rate (complete and partial response) was 18.5%. The clinical benefit rate (complete, partial, and minimal response) was 33.3%. Sixteen patients did not respond to CPT (no change and progressive disease). Patients treated with higher doses of CPT appeared to have better responses., Conclusions: CPT was safe and well tolerated by RRMM patients, and doses between 8 and 15 mg/kg were recommended for the phase 2 study.

Glutamine plays an important role in the metabolism of tumor cells through its contribution to redox homeostasis, bioenergetics, synthesis of macromolecules, and signaling. Triple-negative breast cancers (TNBC) are highly metastatic and associated with poor prognosis. TNBC cells show a marked dependence on extracellular glutamine for growth. Herein we demonstrate that TNBC cells are markedly sensitized to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis upon glutamine deprivation. Upregulation of pro-apoptotic TRAIL receptor 2 (TRAIL-R2/DR5) and downregulation of FLICE-inhibitory protein (FLIP) are observed in glutamine-deprived TNBC cells. Activation of the amino-acid-sensing kinase general control nonderepressible 2 (GCN2) upon glutamine deprivation is responsible for TRAIL-R2 upregulation through a signaling pathway involving ATF4 and CHOP transcription factors. In contrast, FLIP downregulation in glutamine-deprived TNBC occurs by a GCN2-independent mechanism. Importantly, silencing FLIP expression by RNA interference results in a marked sensitization of TNBC cells to TRAIL-induced apoptosis. In addition, pharmacological or genetic inhibition of transaminases increases TRAIL-R2 expression and downregulates FLIP levels, sensitizing TNBC cells to TRAIL. Interestingly, treatment with L-asparaginase markedly sensitizes TNBC cells to TRAIL through its glutaminase activity. Overall, our findings suggest that targeting the glutamine addiction phenotype of TNBC can be regarded as a potential antitumoral target in combination with agonists of proapoptotic TRAIL receptors.

Recent studies suggest that a circulating protein called TRAIL (TNF-related apoptosis inducing ligand) may have an important role in the treatment of type 2 diabetes. It has been shown that TRAIL deficiency worsens diabetes and that TRAIL delivery, when it is given before disease onset, slows down its development. The present study aimed at evaluating whether TRAIL had the potential not only to prevent, but also to treat type 2 diabetes. Thirty male C57BL/6J mice were randomized to a standard or a high-fat diet (HFD). After 4 weeks of HFD, mice were further randomized to receive either placebo or TRAIL, which was delivered weekly for 8 weeks. Body weight, food intake, fasting glucose, and insulin were measured at baseline and every 4 weeks. Tolerance tests were performed before drug randomization and at the end of the study. Tissues were collected for further analyses. Parallel in vitro studies were conducted on HepG2 cells and mouse primary hepatocytes. TRAIL significantly reduced body weight, adipocyte hypertrophy, free fatty acid levels, and inflammation. Moreover, it significantly improved impaired glucose tolerance, and ameliorated non-alcoholic fatty liver disease (NAFLD). TRAIL treatment reduced liver fat content by 47% in vivo as well as by 45% in HepG2 cells and by 39% in primary hepatocytes. This was associated with a significant increase in liver peroxisome proliferator-activated receptor (PPAR) γ (PPARγ) co-activator-1 α (PGC-1α) expression both in vivo and in vitro , pointing to a direct protective effect of TRAIL on the liver. The present study confirms the ability of TRAIL to significantly attenuate diet-induced metabolic abnormalities, and it shows for the first time that TRAIL is effective also when administered after disease onset. In addition, our data shed light on TRAIL therapeutic potential not only against impaired glucose tolerance, but also against NAFLD., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Active targeting nanoparticles were developed to simultaneously codeliver tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Curcumin (Cur). In the nanoparticles (TRAIL-Cur-NPs), TRAIL was used as both active targeting ligand and therapeutic agent, and Cur could upregulate death receptors (DR4 and DR5) to increase the apoptosis-inducing effects of TRAIL. Compared with corresponding free drugs, TRAIL-Cur-NPs group showed enhanced cellular uptake, cytotoxicity and apoptosis induction effect on HCT116 colon cancer cells. In addition, in vivo anticancer studies suggested that TRAIL-Cur-NPs had superior therapeutic effect on tumors without obvious toxicity, which was mainly due to the high tumor targeting and synergistic effect of TRAIL and Cur. The synergistic mechanism of improved antitumor efficacy was proved to be upregulation of DR4 and DR5 in tumor cells induced by Cur. Thus, the prepared codelivery nanoparticles may have potential applications in colorectal cancer therapy.

Although TRAIL (tumor-necrosis-factor (TNF)-related apoptosis-inducing ligand) has been considered a promising broad-spectrum antitumor agent, its further application was limited by poor drug delivery and TRAIL-resistant tumors. A three-step drug delivery strategy was applied to TRAIL for solving these two obstacles in the form of PEG-TRAIL-MMAE (Monomethyl Auristatin E). PEGylation of TRAIL in the first step was carried out to improve its in vivo pharmacokinetics, while the interaction between TRAIL conjugates with death receptors in the second step was designed to activate the TRAIL extrinsic apoptosis pathway, and the further release of MMAE from the lysosome was the third step for introducing another apoptosis pathway to overcome TRAIL resistance in some tumors. Herein, in order to reach a balance among the three steps, the PEG/MMAE ratio was optimized for PEG-TRAIL-MMAE conjugates. PEG-TRAIL-MMAE conjugates with various PEG/MMAE ratios were prepared and compared with each other regarding their pharmacokinetics (PK) and pharmacodynamics (PD). As a result, PEG-TRAIL-MMAE conjugates with a PEG/MMAE ratio of 1:2 showed prolonged half-life in rats (6.8 h), and the best antitumor activity in vitro (IC 50 0.31 nM) and in vivo while no sign of toxicity in xenograft models, suggesting it as a promising multistep drug delivery and antitumor strategy after optimization.

Successful cancer therapies aim to induce selective apoptosis in neoplastic cells. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered an attractive anticancer agent due to its tumor cell-specific cytotoxicity. However, earlier studies with recombinant TRAIL revealed many shortcomings, including a short half-life, off-target toxicity and existence of TRAIL-resistant tumor cells. In this study, we developed a novel engineering strategy for recombinant soluble TRAIL by redesigning its structure with the adenovirus knobless fiber motif to form a stable homotrimer with improved antitumor activity. The result is a highly stable fiber-TRAIL fusion protein that could form homotrimers similar to natural TRAIL. The recombinant fusion TRAIL developed here displayed high specific activity in both cell-based assays in vitro and animal tests in vivo. This construct will serve as a foundation for a new generation of recombinant proteins suitable for use in preclinical and clinical studies and for effective combination therapies to overcome tumor resistance to TRAIL.

Clinical applications of recombinant human tumor necrosis factor-related apoptosis-inducing ligand (hTRAIL) have been limited by their poor pharmacokinetics. Using endogenous albumin as a carrier is an attractive approach for circulatory half-life extension. Here, we produced ABD-hTRAIL and hTRAIL-ABD by fusing the albumin-binding domain (ABD) from protein G to the N- or C-terminus of hTRAIL. We found that ABD-hTRAIL bound human serum albumin (HSA) with a high affinity (0.4 ± 0.18 nM) and formed nanoparticles with an average diameter (~12 nm) above the threshold (~7 nm) of renal filtration. ABD-hTRAIL also bound mouse serum albumin (MSA); thus, its half-life was 40-50-fold greater than that of hTRAIL (14.1 ± 0.87 h vs 0.32 ± 0.14 h). Tumor uptake of ABD-hTRAIL 8-48 h post-injection was 6-16-fold that of hTRAIL. Consequently, the tumor suppression of ABD-hTRAIL in mice bearing subcutaneous xenografts was 3-4 times greater than that of hTRAIL. Additionally, the time period during which ABD-hTRAIL could kill circulating tumor cells was approximately 8 times longer than that of hTRAIL. These results demonstrate that ABD fused to the N-terminus endows hTRAIL with albumin binding ability; once it enters the vasculature, ABD mediates binding with endogenous albumin, thus prolonging the half-life and enhancing the antitumor effect of hTRAIL. However, hTRAIL-ABD did not show a high affinity for albumin and therefore did not display the prolonged circulatory half-life and enhanced antitumor effects. These results demonstrate that N-terminal, but not C-terminal, ABD-fusion is an efficient technique for enhancing the antitumor effects of hTRAIL by using endogenous albumin as a carrier., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) has attracted great interest as a cancer therapy because it selectively induces death receptor (DR)-mediated apoptosis in cancer cells while sparing normal tissue. However, recombinant human TRAIL demonstrates limited therapeutic efficacy in clinical trials, possibly due to TRAIL-resistance of primary cancers and its inherent short half-life. Here we introduce drug delivery approaches to maximize in vivo potency of TRAIL in TRAIL-resistant tumor xenografts by (1) extending the half-life of the ligand with PEGylated TRAIL (TRAILPEG) and (2) concentrating a TRAIL sensitizer, selected from in vitro screening, in tumors via tumor-homing nanoparticles. Antitumor efficacy of TRAILPEG with tumor-homing sensitizer was evaluated in HCT116 and HT-29 colon xenografts. Western blot, real-time PCR, immunohistochemistry and cell viability assays were employed to investigate mechanisms of action and antitumor efficacy of the combination. We discovered that doxorubicin (DOX) sensitizes TRAIL-resistant HT-29 colon cancer cells to TRAIL by upregulating mRNA expression of DR5 by 60% in vitro. Intravenously administered free DOX does not effectively upregulate DR5 in tumor tissues nor demonstrate synergy with TRAILPEG in HT-29 xenografts, but rather introduces significant systemic toxicity. Alternatively, when DOX was encapsulated in hyaluronic acid-based nanoparticles (HAC/DOX) and intravenously administered with TRAILPEG, DR-mediated apoptosis was potentiated in HT-29 tumors by upregulating DR5 protein expression by 70% and initiating both extrinsic and intrinsic apoptotic pathways with reduced systemic toxicity compared to HAC/DOX or free DOX combined with TRAILPEG (80% vs. 40% survival rate; 75% vs. 34% tumor growth inhibition). This study demonstrates a unique approach to overcome TRAIL-based therapy drawbacks using sequential administration of a tumor-homing TRAIL sensitizer and long-acting TRAILPEG., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Poor pharmacokinetics and resistance within some tumor cell lines have been the major obstacles during the preclinical or clinical application of TRAIL (tumor-necrosis-factor (TNF)-related apoptosis-inducing ligand). The half-life of TRAIL114-281 (114 to 281 amino acids) was revealed to be no more than 30 minutes across species. Therefore maleimido activated PEG (polyethylene glycol) and MMAE (Monomethyl Auristatin E) were applied to site-specifically conjugate with the mutated cysteines from different monomers of TRAIL successively, taking advantage of steric effects involved within TRAIL mutant conjugations. As a result, TRAIL trimer was hetero-modified for different purposes. And the resulting PEG-TRAIL-vcMMAE conjugate exhibited dramatically improved half-life (11.54 h), favourable in vivo targeting capability and antitumor activities while no sign of toxicity in xenograft models, suggesting it's a viable therapeutic and drug delivery strategy.

Direct pulmonary delivery of anti-cancer agents is viewed as an effective way of treating lung cancer. Here, we fabricated inhalable nanoparticles made of human serum albumin (HSA) conjugated with doxorubicin and octyl aldehyde and adsorbed with apoptotic TRAIL protein (TRAIL/Dox HSA-NP). The octyl aldehyde and doxorubicin endowed HSA with significant hydrophobicity that facilitated self-assembly. TRAIL/Dox HSA-NP was found to have excellent particle size (~340nm), morphology, dispersability, and aerosolization properties. TRAIL/Dox HSA-NP displayed synergistic cytotoxicity and apoptotic activity in H226 lung cancer cells vs. HSA-NP containing TRAIL or Dox alone. TRAIL/Dox HSA-NP was well deposited in the mouse lungs using an aerosolizer, and TRAIL and Dox-HSA were found to be gradually released over 3days. The anti-tumor efficacy of pulmonary administered TRAIL/Dox HSA-NP was evaluated in BALB/c nu/nu mice bearing H226 cell-induced metastatic tumors. It was found that the tumors of H226-implanted mice treated with TRAIL/Dox HSA-NP were remarkably smaller and lighter than those of mice treated with TRAIL or Dox HSA-NP alone (337.5±7.5; 678.2±51.5; and 598.9±24.8mg, respectively). Importantly, this improved anti-tumor efficacy was found to be due to the synergistic apoptotic effects of Dox and TRAIL. In the authors' opinion, TRAIL/Dox HSA-NP offers a potential inhalable anti-lung cancer drug delivery system. Furthermore, the synergism displayed by combined use of Dox and TRAIL could be used to markedly reduce doxorubicin doses and minimize its side effects., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Albumin conjugation is viewed as an effective means of protracting short in vivo lifespans of proteins and targeting rheumatoid arthritis (RA). In this study, we present a human serum albumin (HSA) conjugate linked with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) via a bifunctional PEG derivative (HSA-TRAIL). Prepared HSA-TRAIL was found to have a larger molecular size (∼240 kDa, 15.4 nm) than TRAIL (∼66 kDa, 6.2 nm), and its bioactivity (apoptosis, cytotoxicity, and antiproliferation) was well preserved in Mia Paca-2 cells and mouse splenocytes. The enhanced therapeutic efficacy of HSA-TRAIL was demonstrated in collagen-induced arthritis (CIA) mice. The incidence and clinical scores, expressed as degree of erythema and swelling in HSA-TRAIL-treated mice, were remarkably lower than those of TRAIL-treated mice. The serum levels of pro-inflammatory cytokines IFN-γ, TNF-α, IL-1β, and IL-2 in HSA-TRAIL-treated mice were significantly lower than those of TRAIL-treated mice. Furthermore, HSA-TRAIL accumulated in the hind paws of CIA mice, not in naïve TRAIL mice. Pharmacokinetic profiles of HSA-TRAIL were greatly improved in comparison to those of TRAIL (AUCinf: 844.1 ± 130.0 vs 36.0 ± 1.2 ng·h/mL; t1/2: 6.20 ± 0.72 vs 0.23 ± 0.01 h, respectively). The HSA-TRAIL conjugate, which presents clear advantages of targeting RA and long systemic circulation by HSA and unique anti-inflammatory efficacy by TRAIL, has potential as a novel treatment for rheumatoid arthritis.

Neuroblastoma (NB), the most common and deadly extracranial solid tumour of childhood, represents a challenging in paediatric oncology. Soluble tumour necrosis factor (TNF)-related apoptosis-inducing ligand (sTRAIL) is a cancer cell-specific molecule exerting remarkable anti-tumour activities against paediatric malignancies both in vitro and in preclinical settings. However, due to its too fast elimination and to the undesired related side effects, the improvement of sTRAIL in vivo bioavailability and the specific delivery to the tumour is mandatory for increasing its therapeutic efficacy. In this manuscript, we developed an innovative pegylated liposomal formulation carrying the sTRAIL at the outer surface (sTRAIL-SL) with the intent to improve its serum half-life and increase its efficacy in vivo, while reducing side effects. Furthermore, the possibility to combine sTRAIL-SL with the proteasome inhibitor Bortezomib (BTZ) was investigated, being BTZ able to sensitize tumour cells toward TRAIL-induced apoptosis. We demonstrated that sTRAIL preserved and improved its anti-tumour activity when coupled to nanocarriers. Moreover, sTRAIL-SL ameliorated its PK profile in blood allowing sTRAIL to exert a more potent anti-tumour activity, which led, upon BTZ priming, to a statistically significant enhanced life spans in two models of sTRAIL-resistant NB-bearing mice. Finally, mechanistic studies indicated that the combination of sTRAIL with BTZ sensitized sTRAIL-resistant NB tumour cells to sTRAIL-induced cell death, both in vitro and in vivo, through the Akt/GSK3/β-catenin axis-dependent mechanism. In conclusion, our results suggest that sTRAIL-SL might be an efficient vehicle for sTRAIL delivery and that its use in clinic, in combination with BTZ, might represent an adjuvant strategy for the treatment of stage IV, sTRAIL-resistant, NB patients., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exhibits potent antitumor activity in a wide range of cancers without deleterious side effects on normal tissues. Several TRAIL derivatives have been developed to improve its pharmacokinetics and therapeutic effects through strategies such as adding a leucine zipper to increase the circulation half-life. To obtain clinical grade LZ-TRAIL for phase I clinical trial, a single batch of 30 L bioreactor culture was performed using the Escherichia coli BL21 (DE3) strain expressing the recombinant LZ-TRAIL. A robust LZ-TRAIL production fermentation process was developed, which could be scaled up from 5L to 50 L, and had a titer of approximately 1.4 g/l. A four-step purification strategy was carried out to obtain a final product with over 95% purity and 45% yield. The final material was filter sterilized, aseptically vialed, and stored at 4°C, and comprehensively characterized using multiple assays (vialed product was sterile, purity was 95%, aggregates were <5%, potency revealed IC50 of 9 nM on MDA-MB-231 cells, and the endotoxin level was <0.25 U/mg). The purity, composition, and functional activities of the molecule were confirmed. in vivo investigations indicated that LZ-TRAIL has better antitumor potency in three Xenograft tumor models compared to TRAIL (95-281). LZ-TRAIL also showed improved pharmacokinetic and safety profiles in cynomolgus monkeys without abnormalities associated with drug exposure. In conclusion, the scalable synthesis of LZ-TRAIL is useful for production of phase I clinical trial material. These preclinical investigations warrant further clinical development of this product for cancer therapy., (Copyright © 2014. Published by Elsevier B.V.)

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising antitumor agent that specifically induces apoptosis in broad-spectrum tumor cell lines, meanwhile leaving normal cells unaffected. Unfortunately, the clinical development of TRAIL was hampered, and could be attributed to its instability, bioavailability or poor delivery. Although N-terminal specific PEGylation provides a means to improve the pharmacokinetic and stability of TRAIL, it took a bit longer time to accomplish the PEGylation process than expected. We therefore designed another PEGylation approach, mutated Cys-SH site-specific PEGylation, to conjugate methoxypoly(ethylene glycol) maleimide (mPEG-MAL) with TRAIL (95-281) mutant N109C. Asn-109 was chosen as the PEGylated site for it is a potential N-linked glycosylation site. It was shown that ~90% TRAIL mutant N109C could be PEGylated by mPEG-MAL within 40 min. And mPEG(MAL)-N109C was revealed to possess superior in vitro stability and antitumor activity than N-terminal specifically PEGylated TRAIL (114-281) (mPEG(ALD)-TRAIL(114-281)). What's more, mPEG(MAL)-N109C exhibited more therapeutic potentials than mPEG(ALD)-TRAIL(114-281) in tumor xenograft model, benefitting from better drug delivery and bioavailability. These results have demonstrated mutated Cys-SH specific PEGylation is an alternative to site-specifically PEGylate TRAIL efficiently and effectively other than N-terminal specific PEGylation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In this issue of The Journal of Investigative Dermatology, Kouno et al. achieve skin-specific drug delivery using an antibody to deliver substances in a highly specific manner to nontransformed cells. They make use of a nonpathogenic anti-desmoglein 3 autoantibody that had been derived from a patient with pemphigus vulgaris to deliver drugs to the surface of keratinocytes. This approach may turn out to be a new "magic bullet", thereby revolutionizing the therapy of skin disease. The authors then used a conjugate of this antibody with a new drug entity, TNF-related apoptosis-inducing ligand, to demonstrate, as a proof-of-principle, that their approach has the potential to facilitate the treatment of both cancerous and inflammatory skin diseases.

We determined the feasibility of using an anti-desmoglein (Dsg) mAb, Px44, to deliver a biologically active protein to keratinocytes. Recombinantly produced Px44-green fluorescent protein (GFP) injected into mice and skin organ culture delivered GFP to the cell surface of keratinocytes. We replaced GFP with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to produce Px44-TRAIL. We chose TRAIL as a biological model because it inhibits activated lymphocytes and causes apoptosis of hyperproliferative keratinocytes, features of various skin diseases. Px44-TRAIL formed a trimer, the biologically active form of TRAIL. Standard assays of TRAIL activity showed that Px44-TRAIL caused apoptosis of Jurkat cells and inhibited IFN-γ production by activated CD4+ T cells. Enzyme-linked immunoassay with Px44-TRAIL showed delivery of TRAIL to Dsg. Immunofluorescence with Px44-TRAIL incubated on skin sections and cultured keratinocytes or injected into mouse skin, human organ culture, or human xenografts detected TRAIL on keratinocytes. Px44-TRAIL caused apoptosis of the hyperproliferative, but not differentiating, cultured keratinocytes through binding to Dsg3. Foldon, a small trimerization domain, cloned into Px44-TRAIL maintained its stability and biological activity at 37° C for at least 48 hours. These data suggest that such targeted therapy is feasible and may be useful for hyperproliferative and inflamed skin diseases.

Transferrin (Tf) is considered an effective tumor-targeting agent, and PEGylation effectively prolongs in vivo pharmacokinetics by delaying excretion via the renal route. The authors describe the active tumor targeting of long-acting Tf-PEG-TNF-related apoptosis-inducing ligand conjugate (Tf-PEG-TRAIL) for effective cancer therapy. Tf-PEG-TRAIL was prepared using a two-step N-terminal specific PEGylation procedure using different PEGs (Mw: 3.4, 5, 10 kDa). Eventually, only 10 kDa PEG was linked to Tf and TRAIL because TRAIL (66 kDa) and Tf (81 kDa) were too large to link to 3.4 and 5 kDa PEG. The final conjugate Tf-PEG(10K)-TRAIL was successfully purified and characterized by SDS-PAGE, western blotting. To determine the specific binding of Tf-PEG(10K)-TRAIL to Tf receptor, competitive receptor binding assays were performed on K 562 cells. The results obtained demonstrate that the affinity of Tf-PEG(10K)-TRAIL for Tf receptor is similar to that of native Tf. In contrast, PEG(10K)-TRAIL demonstrated no specificity. Biodistribution patterns and antitumor effects were investigated in C57BL6 mice bearing B16F10 murine melanomas and BALB/c athymic mice bearing HCT116. Tumor accumulation of Tf-PEG(10K)-TRAIL was 5.2 fold higher (at 2 h) than TRAIL, because Tf-PEG(10K)-TRAIL has both passive and active tumor targeting ability. Furthermore, the suppression of tumors by Tf-PEG(10K)-TRAIL was 3.6 and 1.5 fold those of TRAIL and PEG(10K)-TRAIL, respectively. These results suggest that Tf-PEG(10K)-TRAIL is a superior pharmacokinetic conjugate that potently targets tumors and that it should be viewed as a potential cancer therapy., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Combination chemotherapy represents the standard-of-care for non-Hodgkin lymphoma. However, the development of new therapeutic regimens is empirical and this approach cannot be used prospectively to identify novel or optimal drug combinations. Quantitative system pharmacodynamic models could promote the discovery and development of combination regimens based upon first principles. In this study, we developed a mathematical model that integrates temporal patterns of drug exposure, receptor occupancy, and signal transduction to predict the effects of the CD20 agonist rituximab in combination with rhApo2L/TNF-related apoptosis inducing ligand or fenretinide, a cytotoxic retinoid, upon growth kinetics in non-Hodgkin lymphoma xenografts. The model recapitulated major regulatory mechanisms, including target-mediated disposition of rituximab, modulation of proapoptotic intracellular signaling induced by CD20 occupancy, and the relative efficacy of death receptor isoforms. The multiscale model coupled tumor responses to individual anticancer agents with their mechanisms of action in vivo, and the changes in Bcl-xL and Fas induced by CD20 occupancy were linked to explain the synergy of these drugs. Tumor growth profiles predicted by the model agreed with cell and xenograft data, capturing the apparent pharmacologic synergy of these agents with fidelity. Together, our findings provide a mechanism-based platform for exploring new regimens with CD20 agonists., (©2012 AACR.)

Background and Purpose: The TNF-related apoptosis inducing ligand (TRAIL) induces apoptosis through activation of the death receptors, TRAIL-R1 and TRAIL-R2. Recombinant human (rh) TRAIL and the TRAIL-R1 directed monoclonal antibody mapatumumab are currently clinically evaluated as anticancer agents. The objective of this study was to develop radiopharmaceuticals targeting the TRAIL-R1, suitable for clinical use to help understand and predict clinical efficacy in patients., Experimental Approach: rhTRAIL was radioiodinated with (125) I, and conjugated mapatumumab was radiolabelled with (111) In. The radiopharmaceuticals were characterized, their in vitro stability and death receptor targeting capacities were determined and in vivo biodistribution was studied in nude mice bearing human tumour xenografts with different expression of TRAIL-R1., Key Results: Labelling efficiencies, radiochemical purity, stability and binding properties were optimized for the radioimmunoconjugates. In vivo biodistribution showed rapid renal clearance of [(125) I]rhTRAIL, with highest kidney activity at 15 min and almost no detectable activity after 4 h. Activity rapidly decreased in almost all organs, except for the xenografts. Radiolabelled mapatumumab showed blood clearance between 24 and 168 h and a reduced decrease in radioactivity in the high receptor expression xenograft., Conclusions and Implications: rhTRAIL and mapatumumab can be efficiently radiolabelled. The new radiopharmaceuticals can be used clinically to study pharmacokinetics, biodistribution and tumour targeting, which could support evaluation of the native targeted agents in phase I/II trials., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Malignant glioma is the most frequent type in brain tumors. The prognosis of this tumor has not been significantly improved for the past decades and the average survival of patients is less than one year. Thus, an effective novel therapy is urgently needed. TNF-related apoptosis inducing ligand (TRAIL), known to have tumor cell-specific killing activity, has been investigated as a novel therapeutic for cancers. We have developed Ad-stTRAIL, an adenovirus delivering secretable trimeric TRAIL for gene therapy and demonstrated the potential to treat malignant gliomas. Currently, this Ad-stTRAIL gene therapy is under phase I clinical trial for malignant gliomas. Here, we report preclinical studies for Ad-stTRAIL carried out using rats. We delivered Ad-stTRAIL intracranially and determined its pharmacokinetics and biodistribution. Most Ad-stTRAIL remained in the delivered site and the relatively low number of viral genomes was detected in the opposite site of brain and cerebrospinal fluid. Similarly, only small portion of the viral particles injected was found in the blood plasma and major organs and tissues, probably due to the brain-blood barrier. Multiple administrations did not lead to accumulation of Ad-stTRAIL at the injection site and organs. Repeated delivery of Ad-stTRAIL did not show any serious side effects. Our data indicate that intracranially delivered Ad-stTRAIL is a safe approach, demonstrating the potential as a novel therapy for treating gliomas.

TRAIL has received considerable attention as a potential anti-cancer agent due to its specific ability to target tumors. However, recombinant TRAIL has several limitations, such as, its short biological half-life, its inherent instability, and its potential hepatotoxicity. In this study, we developed a sustained release nanoparticle formulation of TRAIL and investigated its therapeutic effects in tumor-bearing mice. TRAIL-loaded nanoparticles (NPs) were prepared by mixing PEGylated heparin (PEG-HE), poly-L-lysine (PLL), and TRAIL. NPs prepared by the ionic interaction between polymer and TRAIL showed uniform spherical structures of diameter 213.3 ± 9.7 nm and a surface charge of 5.33 ± 1.2 mV. An in vitro study of the bioactivity of TRAIL in NPs showed that TRAIL-loaded PEG-HE/PLL NPs (TRAIL-PEG-NPs) were slightly less cytotoxic than TRAIL in vitro. To investigate pharmacokinetic parameters, TRAIL and TRAIL-PEG-NPs were intravenously injected into SD rats. The PEG-NP-based formulation demonstrated a 28.3 fold greater half-life than TRAIL alone. To evaluate the anti-tumor effect, TRAIL, TRAIL-loaded HE/PLL NPs (TRAIL-NPs), and TRAIL-PEG-NPs were intravenously injected into HCT-116 tumor-bearing BALB/c athymic mice. The TRAIL-PEG-NP formulation efficiently suppressed tumor growth (>70%), and histological findings confirmed that NPs induced significant tumor cell apoptosis without inducing liver toxicity. The PEG-exposed NP fabrication method applied in this study could be widely applied to protein and peptide delivery systems., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The purpose of this work was to develop an effective PEGylated TNF-related apoptosis-inducing ligand (PEG-TRAIL) delivery system for antitumor therapy based on local injection to tumor sites that has a sustained effect without protein aggregation or an initial release burst. The authors designed poly (lactic-co-glycolic) acid (PLGA) microspheres that deliver PEG-TRAIL locally and continuously at tumor sites with sustained biological activity and compared its performance with that of TRAIL microspheres. TRAIL or PEG-TRAIL was microencapsulated into PLGA microspheres using a double-emulsion solvent extraction method. Prepared TRAIL and PEG-TRAIL microspheres showed entirely spherical, smooth surfaces. However, PEG-TRAIL microspheres exhibited a 2.07-fold higher encapsulation efficiency than TRAIL microspheres, and exhibited a tri-phasic in vitro release profile with a lower initial burst (15.8%) than TRAIL microspheres (42.7%). Furthermore, released PEG-TRAIL showed a continued ability to induce apoptosis over 14 days. In vivo pharmacokinetic studies also demonstrated that PEG-TRAIL microspheres had a sustained release profile (18 days), and that the steady-state concentration of PEG-TRAIL in rat plasma was reached at day 3 and maintained until day 15; its steady-state concentration in rat plasma changed from 1444.3 ± 338.4 to 2697.7 ± 419.7 pg/ml. However, TRAIL microspheres were released out within 2 days after administration. Finally, in vivo antitumor tests revealed that tumor growths were significantly more inhibited by a single dose of PEG-TRAIL microspheres than TRAIL microspheres when delivered at 300 μg of TRAIL/mouse. Tumors taken from mouse treated with PEG-TRAIL microspheres showed 78.3% tumor suppression at 24 days, and this was 3.02-fold higher than that observed for TRAIL microspheres (25.9% tumor inhibition). Furthermore, these improved pharmaceutical characteristics of PEG-TRAIL microspheres resulted in superior therapeutic effects without detectable side effects. These findings strongly suggest that PEG-TRAIL microspheres offer a new therapeutic strategy for the treatment of cancers., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has received considerable attention as a potential anticancer agent. However, recombinant Apo2L/TRAIL has several limitations, which include a weak pharmacokinetic profile, namely, a short biological half-life and rapid renal clearance, and an inability to form a homotrimeric structure. In this research, we attempted to develop a sustained release nanoparticle (NP) formulation that stabilizes Apo2L/TRAIL and preserves its antitumor activity. Apo2L/TRAIL-loaded human serum albumin (HSA) NPs were prepared using a desolvation technique optimized by particle size, zeta-potential, and entrapment efficiency. Apo2L/TRAIL in HSA-NPs continuously released over 24 h at 37°C in phosphate buffered saline and rat plasma condition, and the biological activity of Apo2L/TRAIL-HSA-NPs was preserved (IC(50) = 67.2 ng/mL versus Apo2L/TRAIL IC(50) = 55.4 ng/mL) with negligible activity loss. Furthermore, in vivo pharmacokinetic profiles and tumor distribution demonstrated the superiority of Apo2L/TRAIL-HSA-NPs over Apo2L/TRAIL. The circulating half-life period was significantly prolonged from 9.8 to 90.7 min (9.2-fold enhancement), and drug bioavailability was clearly enhanced on the basis of area under the curve analysis (2.7-fold). And tumor distribution of Apo2L/TRAIL-HSA-NPs was also increased at 1 h after injection, which was about 14-fold (1-h point) over that of Apo2L/TRAIL. These results show that Apo2L/TRAIL-loaded HSA-NPs should be considered as potential long-acting cancer agents., (Copyright © 2010 Wiley-Liss, Inc.)

The clinical applications of tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL), an emerging therapeutic protein for cancer and rheumatoid arthritis (RA), are limited by its instability and short biological half-life. In this study, efficient therapeutic modalities for RA treatment were developed in the form of nano-sized complexes (nanocomplexes) based on hyaluronic acid (HA) and polyethylene glycol (PEG)-derivatized TRAIL (PEG-TRAIL) formed by N-terminal specific PEGylation. The nanocomplexes were prepared by simply mixing the positively charged PEG-TRAIL and negatively charged HA, and showed negligible loss of bioactivity compared with the PEG-TRAIL. The in vivo biodistribution and diffusion kinetics of Cy5.5-labeled PEG-TRAIL in mice were observed using a near-infrared optical imaging system after subcutaneous injection of three different formulations: PEG-TRAIL in phosphate-buffered saline (PBS, pH 7.4), nanocomplex in PBS, or nanocomplex in 1% HA solution. The results suggested that PEG-TRAIL is released slowly in vivo from the nanocomplex in 1% HA. Experiments in a collagen-induced arthritis mouse model demonstrated that the magnitudes of therapeutic effects, as judged by clinical scores and histology, were significantly enhanced by the sustained delivery of PEG-TRAIL, with the order of nanocomplex in 1% HA>nanocomplex in PBS>PEG-TRAIL in PBS. In addition, sustained delivery of PEG-TRAIL from the nanocomplex in 1% HA resulted in significant reduction of serum inflammatory cytokines and collagen-specific antibodies that are responsible for the pathogenesis of RA. These results imply that HA/PEG-TRAIL nanocomplex formulations are promising therapeutic modalities for the treatment of RA., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

In an attempt to improve TRAIL's (tumor necrosis factor-related apoptosis-inducing ligand) tumor selective activity a variant was designed, in which the three TRAIL protomers are expressed as a single polypeptide chain (scTRAIL). By genetic fusion with a single-chain antibody fragment (scFv) recognizing the extracellular domain of ErbB2, we further equipped scTRAIL with tumor-targeting properties. We studied tumor targeting and apoptosis induction of scFv-scTRAIL in comparison with non-targeted scTRAIL. Importantly, the tumor antigen-targeted scTRAIL fusion protein showed higher apoptotic activity in vitro, with a predominant action by TRAIL-R2 signaling. Pharmacokinetic studies revealed increased plasma half-life of the targeted scTRAIL fusion protein compared with scTRAIL. In vivo studies in a mouse tumor model with xenotransplanted Colo205 cells confirmed greater response to the ErbB2-specific scTRAIL fusion protein compared with non-targeted scTRAIL both under local and systemic application regimen. Together, in vitro and in vivo data give proof of concept of higher therapeutic activity of tumor-targeted scFv-scTRAIL molecules. Further, we envisage that through targeting of scTRAIL, potential side effects should be minimized. We propose that scFv-mediated tumor targeting of single-chain TRAIL represents a promising strategy to improve TRAIL's antitumoral action and to minimize potential unwanted actions on normal tissues.Cell Death and Disease (2010) 1, e68; doi:10.1038/cddis.2010.45; published online 26 August 2010.

Purpose: Apoptosis ligand 2/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL)-a member of the tumor necrosis factor cytokine family-induces apoptosis by activating the extrinsic pathway through the proapoptotic death receptors DR4 and DR5. Recombinant human Apo2L/TRAIL (rhApo2L/TRAIL) has broad potential as a cancer therapy. To the best of our knowledge, this is the first in-human clinical trial to assess the safety, tolerability, pharmacokinetics, and antitumor activity of multiple intravenous doses of rhApo2L/TRAIL in patients with advanced cancer., Patients and Methods: This phase I, open-label, dose-escalation study treated patients with advanced cancer with rhApo2L/TRAIL doses ranging from 0.5 to 30 mg/kg/d, with parallel dose escalation for patients without liver metastases and with normal liver function (cohort 1) and for patients with liver metastases and normal or mildly abnormal liver function (cohort 2). Doses were given daily for 5 days, with cycles repeating every 3 weeks. Assessments included adverse events (AEs), laboratory tests, pharmacokinetics, and imaging to evaluate antitumor activity., Results: Seventy-one patients received a mean of 18.3 doses; seven patients completed all eight treatment cycles. The AE profile of rhApo2L/TRAIL was similar in cohorts 1 and 2. The most common AEs were fatigue (38%), nausea (28%), vomiting (23%), fever (23%), anemia (18%), and constipation (18%). Liver enzyme elevations were concurrent with progressive metastatic liver disease. Two patients with sarcoma (synovial and undifferentiated) experienced serious AEs associated with rapid tumor necrosis. Two patients with chondrosarcoma experienced durable partial responses to rhApo2L/TRAIL., Conclusion: At the tested schedule and dose range, rhApo2L/TRAIL was safe and well tolerated. Dose escalation achieved peak rhApo2L/TRAIL serum concentrations equivalent to those associated with preclinical antitumor efficacy.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered an attractive anticancer agent due to its tumor cell-specific cytotoxicity. However, its low stability, solubility, unexpected side effects, and weak pharmacokinetic profiles restrict its successful clinical application. To develop efficient TRAIL-based anticancer biotherapeutics, a new version of trimeric TRAIL was constructed by incorporating trimer-forming zipper sequences (HZ-TRAIL), and then NH(2)-terminal-specific PEGylation was done to produce PEGylated TRAIL (PEG-HZ-TRAIL). The biological, physicochemical, and pharmaceutical characteristics of PEG-HZ-TRAIL were then investigated using various in vitro and in vivo experiments, including a cell-based cytotoxicity test, a solubility test, pharmacokinetic analysis, and antitumor efficacy evaluations. Although slight activity loss occurred after PEGylation, PEG-HZ-TRAIL showed excellent tumor cell-specific cytotoxic effects via apoptotic pathways with negligible normal cell toxicity. The stability and pharmacokinetic problems of HZ-TRAIL were successfully overcome by PEGylation. Furthermore, in vivo antitumor tests revealed that PEG-HZ-TRAIL treatment enhanced therapeutic potentials compared with HZ-TRAIL in tumor xenograft animal models, and these enhancements were attributed to its better pharmacokinetic properties and tumor-targeting performance. These findings show that PEG-HZ-TRAIL administration provides an effective antitumor treatment, which exhibits superior tumor targeting and better inhibits tumor growth, and suggest that PEG-HZ-TRAIL should be considered a potential candidate for antitumor biotherapy.

Purpose: Recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL) is clinically evaluated as novel anticancer drug. rhTRAIL-DR5, a rhTRAIL variant that specifically binds to DR5 receptor, has recently been developed. We investigated whether rhTRAIL-DR5 is more efficient than rhTRAIL in combination with cisplatin in DR5-expressing human A2780 ovarian cancer cells., Design: Effect of cisplatin alone or in combination with rhTRAIL or rhTRAIL-DR5 on DR5 surface expression, apoptosis, and cell survival of A2780 was measured. Biodistribution analysis was done in mice with (125)I-rhTRAIL administered intravenously versus intraperitoneally. Antitumor efficacy of rhTRAIL-DR5 versus rhTRAIL was determined in an intraperitoneally growing bioluminescent A2780 xenograft model., Results: Cisplatin strongly enhanced DR5 surface expression. Both rhTRAIL and rhTRAIL-DR5 in combination with cisplatin induced high levels of caspase-3 activation, apoptosis, and cell kill, with rhTRAIL-DR5 being most potent. Intraperitoneal administration of (125)I-rhTRAIL resulted in a 1.7-fold higher area under the curve in serum, increased tumor exposure, and more caspase-3 activation in the tumor than intravenous administration. Intraperitoneal administration of rhTRAIL-DR5 delayed A2780 tumor progression, reflected in a mean light reduction of 68.3% (P = 0.015), whereas rhTRAIL or rhTRAIL-DR5 plus cisplatin resulted in 85% (P = 0.003) and 97% (P = 0.002) reduction compared with A2780 tumor progression in vehicle-treated animals. Combination of rhTRAIL-DR5 with cisplatin was more effective than cisplatin alone (P = 0.027)., Conclusion: rhTRAIL-DR5 was superior over rhTRAIL in vitro and in vivo against DR5-expressing ovarian cancer also in combination with cisplatin. Intraperitoneal administration of rhTRAIL-DR5 warrants further exploration in ovarian cancer.

The tumor necrosis factor-related apoptosis inducing ligand (TRAIL) has gained much attention due to its potent therapeutic effect for cancer and rheumatoid arthritis. In this study, we attempted to develop the injectable formulations which can stabilize TRAIL and thus show prolonged blood circulation in vivo. The positively charged TRAIL was mixed with hyaluronic acid (HA), resulting in the formation of nanocomplexes. The zeta-potentials of nanocomplexes and their mean diameters were significantly dependent on the feed ratio of HA to TRAIL. The increase in the feed ratio of HA reduced the particle size and decreased the value of the zeta-potential. The bioactivity of TRAIL in the complexes was comparable to that of native TRAIL, indicating that the complex formation did not affect the activity of TRAIL. Furthermore, the stability of TRAIL in the complexes was retained for 6 days, during which the bioactivity of native TRAIL disappeared. When native TRAIL was subcutaneously injected into the rats, its plasma concentration was not detectable after 12h. In contrast, for HA/TRAIL nanocomplexes in 1% HA solution, substantial amount of TRAIL was circulated in blood for up to 5 days. These results imply that HA-based formulations of TRAIL hold the potential as the therapeutics.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent with tumor-selective apoptotic activity. TRAIL plays a role in the innate and adaptive immune response and autoimmune disease and may also be involved in hepatic cell death and inflammation. For these reasons, chronic exposure to TRAIL may have deleterious side effects in patients as a cancer therapeutic. In this study, we have improved the antitumor activity of TRAIL by targeted delivery to the tumor vasculature, leading to dramatic enhancement of its therapeutic properties. TRAIL was fused to the ACDCRGDCFC peptide (named RGD-L-TRAIL), a ligand of alpha(V)beta(3) and alpha(V)beta(5) integrins. Biological activity was evaluated in vitro and antitumor efficacy was investigated in vivo as a single agent and in combination with irinotecan hydrochloride (CPT-11). The fusion protein RGD-L-TRAIL, but not TRAIL or RGE-L-TRAIL, specifically bound to microvascular endothelial cells in a dose-dependent manner and showed enhanced apoptosis-inducing activity (caspase-3 and caspase-8 activation) in alpha(V)beta(3) and alpha(V)beta(5) integrin-positive cancer cells. In addition, RGD-L-TRAIL was more effective in suppressing tumor growth of COLO-205 tumor-bearing mice than an equivalent dose of TRAIL. The antitumor effect of RGD-L-TRAIL was further enhanced by combination with CPT-11 in both TRAIL-sensitive COLO-205 and TRAIL-resistive HT-29 tumor xenograft models. Our findings suggest that the novel fusion protein RGD-L-TRAIL can directly target tumor endothelial cells as well as alpha(V)beta(3) and alpha(V)beta(5) integrin-positive tumor cells. The tumor-targeted delivery of TRAIL derivatives, such as RGD-L-TRAIL, may prove to be a promising lead candidate for cancer therapy.

TRAIL is a potent antitumor agent, but its potential toxicity to normal human tissues limits its clinical applications in future. Therapy of human tumors might benefit from the use of vectors enabling tight control of TRAIL expression in vivo. To this aim, we constructed an adenoviral vector carrying the RU486-dependent gene switch system for the regulable expression of recombinant TRAIL. Only was apoptotic recombinant TRAIL expressed and cytotoxicity observed upon binding of RU 486 to the inducible promoter. Expression levels and kinetics of recombinant TRAIL expression could be achieved by modulating the concentration of the inducer. As a broad implication, our data provide an alternative approach to circumvent the potential toxicity of TRAIL in future human trials and this system may be utilized to treat human cancer using a long-term expression vector.

Patients with malignant gliomas have a poor prognosis because these tumors do not respond well to conventional treatments. Studies of glioma xenografts suggest that they may be amenable to gene therapy with cytotoxic genes, such as the proapoptotic Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL). Gene therapy of gliomas ideally employs i.v. given vectors, thus excluding viral vectors as they cannot cross the brain microvascular endothelium or blood-brain barrier. Recently, we reported the synthesis of cationic albumin-conjugated pegylated nanoparticles (CBSA-NP) and showed their accumulation in mouse brain cells upon i.v. administration. In this study, plasmid pORF-hTRAIL (pDNA) was incorporated into CBSA-NP, and the resulting CBSA-NP-hTRAIL was evaluated as a nonviral vector for gene therapy of gliomas. Thirty minutes after transfection of C6 glioma cells, CBSA-NP-hTRAIL was internalized and mostly located in the cytoplasm, whereas NP-hTRAIL was entrapped in the endolysosomal compartment. At 6 and 48 hours after transfection, respectively, released pDNA was present in the nuclei and induced apoptosis. At 30 minutes after i.v. administration of CBSA-NP-hTRAIL to BALB/c mice bearing i.c. C6 gliomas, CBSA-NP-hTRAIL colocalized with glycoproteins in brain and tumor microvasculature and, via absorptive-mediated transcytosis, accumulated in tumor cells. At 24 and 48 hours after i.v. administration of CBSA-NP-hTRAIL, respectively, hTRAIL mRNA and protein were detected in normal brain and tumors. Furthermore, repeated i.v. injections of CBSA-NP-hTRAIL induced apoptosis in vivo and significantly delayed tumor growth. In summary, this study indicates that CBSA-NP-hTRAIL is a promising candidate for noninvasive gene therapy of malignant glioma.

Antiviral treatment with pegylated interferon (PEG-IFN) and ribavirin for chronic hepatitis C improved the rates of viral clearance to 56%. An open issue is a better understanding of the factors responsible for the residual lack of response. Our aim was to investigate the effect of antiviral treatments on soluble tumor necrosis factor-related ligand (sTRAIL), which is capable of inducing apoptosis in virus-infected cells. We analyzed sTRAIL levels in 22 naive patients, randomly assigned to receive 6 months of treatment with IFN alone or in combination with amantadine or ribavirin, at baseline, at 6, 12, 24, 30, and 48 h, at days 3, 7, and 14, at 1, 3, and 6 months of treatment, and finally 6 months after the end of treatment. At baseline, the sTRAIL level was significantly higher in patients than in controls (p < 0.0001). The highest sTRAIL release was obtained within the first 12 h, followed by a second peak after the second dose of IFN. There was then a slow decline within the first month. Compared with baseline, high sTRAIL levels were present till day 7 in sustained responders (7 patients) and till the third month of treatment in relapsers or nonresponders (15 patients) (p < 0.02), with no differences related to the type of treatment. The IFN effect on sTRAIL is rapid and intense. The overexpression of TRAIL in viral hepatitis could be seen as a defense mechanism to eliminate infected cells and limit viral replication.