TRAIL reduces impaired glucose tolerance and NAFLD in the high-fat diet fed mouse.

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.

Optimizing Multistep Delivery of PEGylated Tumor-Necrosis-Factor-Related Apoptosis-Inducing Ligand-Toxin Conjugates for Improved Antitumor Activities.

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 (IC50 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.

Human serum albumin-TRAIL conjugate for the treatment of rheumatoid arthritis.

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-1ß, 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.

Enhanced antitumor efficacy of a DR5-specific TRAIL variant over recombinant human TRAIL in a bioluminescent ovarian cancer xenograft model.

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.

Enhancement of antitumor properties of TRAIL by targeted delivery to the tumor neovasculature.

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.

Stability and bioactivity of nanocomplex of TNF-related apoptosis-inducing ligand.

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.

Cationic albumin-conjugated pegylated nanoparticles allow gene delivery into brain tumors via intravenous administration.

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.

A Phase1b Dose Escalation Study of Recombinant Circularly Permuted TRAIL in Patients With Relapsed or Refractory Multiple Myeloma.

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.

Exosomes derived from TRAIL-engineered mesenchymal stem cells with effective anti-tumor activity in a mouse melanoma model.

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].

Glutamine metabolism regulates FLIP expression and sensitivity to TRAIL in triple-negative breast cancer cells.

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.

TRAIL and curcumin codelivery nanoparticles enhance TRAIL-induced apoptosis through upregulation of death receptors.

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.

Soluble tumor necrosis factor-related ligand (sTRAIL) levels and kinetics during antiviral treatment in chronic hepatitis C.

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.

Fusion to an albumin-binding domain with a high affinity for albumin extends the circulatory half-life and enhances the in vivo antitumor effects of human 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.

Engineered adenovirus fiber shaft fusion homotrimer of soluble TRAIL with enhanced stability and antitumor activity.

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.

Inhalable self-assembled albumin nanoparticles for treating drug-resistant lung cancer.

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.

Delivery of tumor-homing TRAIL sensitizer with long-acting TRAIL as a therapy for TRAIL-resistant tumors.

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.

Hetero-modification of TRAIL trimer for improved drug delivery and in vivo antitumor activities.

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.

sTRAIL coupled to liposomes improves its pharmacokinetic profile and overcomes neuroblastoma tumour resistance in combination with Bortezomib.

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/ß-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.

GMP production and characterization of leucine zipper-tagged tumor necrosis factor-related apoptosis-inducing ligand (LZ-TRAIL) for phase I clinical trial.

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.

Skin-specific drug delivery: a rapid solution to skin diseases?

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.