Clinical potential of microdystrophin as a surrogate endpoint.

Accelerated approval based on a likely surrogate endpoint can be life-changing for patients suffering from a rare progressive disease with unmet medical need, as it substantially hastens access to potentially lifesaving therapies. In one such example, antisense morpholinos were approved to treat Duchenne muscular dystrophy (DMD) based on measurement of shortened dystrophin in skeletal muscle biopsies as a surrogate biomarker. New, promising therapeutics for DMD include AAV gene therapy to restore another form of dystrophin termed mini- or microdystrophin. AAV-microdystrophins are currently in clinical trials but have yet to be accepted by regulatory agencies as reasonably likely surrogate endpoints. To evaluate microdystrophin expression as a reasonably likely surrogate endpoint for DMD, this review highlights dystrophin biology in the context of functional and clinical benefit to support the argument that microdystrophin proteins have a high probability of providing clinical benefit based on their rational design. Unlike exon-skipping based strategies, the approach of rational design allows for functional capabilities (i.e. quality) of the protein to be maximized with every patient receiving the same optimized microdystrophin. Therefore, the presence of rationally designed microdystrophin in a muscle biopsy is likely to predict clinical benefit and is consequently a strong candidate for a surrogate endpoint analysis to support accelerated approval.

First-in-human study of the PARP/tankyrase inhibitor E7449 in patients with advanced solid tumours and evaluation of a novel drug-response predictor.

BACKGROUND: This phase 1 study examined the safety, maximum-tolerated dose (MTD) and antitumour activity of E7449, a novel PARP 1/2 and tankyrase 1/2 inhibitor. METHODS: E7449 was orally administered once daily in 28-day cycles to patients with advanced solid tumours (50-800-mg doses). Archival tumour samples from consenting patients were evaluated for the expression of 414 genes in a biomarker panel (2X-121 drug-response predictor [DRP]) found to be predictive of the response to E7449 in cell lines. RESULTS: Forty-one patients were enrolled (13 pancreatic, 5 ovarian, 4 each with breast, lung or colorectal cancer and 11 with other tumour types). The most common grade ≥3 treatment-related adverse event was fatigue (n = 7, 17.1%). Five patients experienced a dose-limiting toxicity (fatigue, n = 4, 800 mg; anaphylaxis, n = 1, 600 mg) for an MTD of 600 mg. E7449 exhibited antitumour activity in solid tumours, including 2 partial responses (PRs), and stable disease (SD) in 13 patients, which was durable (>23 weeks) for 8 patients. In 13 patients, the 2X-121 DRP identified those achieving PR and durable SD. E7449 showed good tolerability, promising antitumour activity and significant concentration-dependent PARP inhibition following 50-800-mg oral dosing. CONCLUSION: The results support further clinical investigation of E7449 and its associated biomarker 2X-121 DRP. CLINICAL TRIAL REGISTRATION: www.ClinicalTrials.gov code: NCT01618136.

Neuropilin-1 drives tumor-specific uptake of chlorotoxin.

BACKGROUND: Chlorotoxin (Cltx) isolated from scorpion venom is an established tumor targeting and antiangiogenic peptide. Radiolabeled Cltx therapeutic (131I-TM601) yielded promising results in human glioma clinical studies, and the imaging agent tozuleristide, is under investigation in CNS cancer studies. Several binding targets have previously been proposed for Cltx but none effectively explain its pleiotropic effects; its true target remains ambiguous and is the focus of this study. METHODS: A peptide-drug conjugate (ER-472) composed of Cltx linked to cryptophycin as warhead was developed as a tool to probe the molecular target and mechanism of action of Cltx, using multiple xenograft models. RESULTS: Neuropilin-1 (NRP1), an endocytic receptor on tumor and endothelial cells, was identified as a novel Cltx target, and NRP1 binding by Cltx increased drug uptake into tumor. Metabolism of Cltx to peptide bearing free C-terminal arginine, a prerequisite for NRP1 binding, took place in the tumor microenvironment, while native scorpion Cltx with amidated C-terminal arginine did not bind NRP1, and instead acts as a cryptic peptide. Antitumor activity of ER-472 in xenografts correlated to tumor NRP1 expression. Potency was significantly reduced by treatment with NRP1 blocking antibodies or knockout in tumor cells, confirming a role for NRP1-binding in ER-472 activity. Higher cryptophycin metabolite levels were measured in NRP1-expressing tumors, evidence of NRP1-mediated enhanced drug uptake and presumably responsible for the superior antitumor efficacy. CONCLUSIONS: NRP1 was identified as a novel Cltx target which enhances tumor drug uptake. This finding should facilitate tumor selection for chlorotoxin-based therapeutics and diagnostics.

Broad-spectrum Preclinical Antitumor Activity of Eribulin (Halaven®): Combination with Anticancer Agents of Differing Mechanisms.

BACKGROUND: Eribulin is used in many countries to treat patients with advanced breast cancer or liposarcoma and exerts in vivo anticancer activity under monotherapy conditions against various human tumor xenograft models. Here, eribulin in combination with mechanistically different anticancer agents was evaluated. MATERIALS AND METHODS: Eribulin was combined with cytotoxic agents (capecitabine, carboplatin, cisplatin, doxorubicin, gemcitabine) or targeted agents (bevacizumab, BKM-120, E7449, erlotinib, everolimus, lenvatinib, palbociclib) in tumor xenograft models of breast cancer, melanoma, non-small cell lung cancer (NSCLC), and ovarian cancer. RESULTS: Across nearly all models, eribulin with either cytotoxic or targeted agents demonstrated combination activity, defined as the activity demonstrably greater than that of either agent alone. Combination activity was absent only with doxorubicin (MDA-MB-435 model) and with lenvatinib (NCI-H1975 model), both of which responded to the agents as monotherapy. CONCLUSION: Eribulin has combination activity with multiple agents from different mechanistic classes in several human cancer models, including breast, NSCLC, ovarian, and melanoma.

E7449: A dual inhibitor of PARP1/2 and tankyrase1/2 inhibits growth of DNA repair deficient tumors and antagonizes Wnt signaling.

Inhibition of Poly(ADP-ribose) Polymerase1 (PARP1) impairs DNA damage repair, and early generation PARP1/2 inhibitors (olaparib, niraparib, etc.) have demonstrated clinical proof of concept for cancer treatment. Here, we describe the development of the novel PARP inhibitor E7449, a potent PARP1/2 inhibitor that also inhibits PARP5a/5b, otherwise known as tankyrase1 and 2 (TNKS1 and 2), important regulators of canonical Wnt/ß-catenin signaling. E7449 inhibits PARP enzymatic activity and additionally traps PARP1 onto damaged DNA; a mechanism previously shown to augment cytotoxicity. Cells deficient in DNA repair pathways beyond homologous recombination were sensitive to E7449 treatment. Chemotherapy was potentiated by E7449 and single agent had significant antitumor activity in BRCA-deficient xenografts. Additionally, E7449 inhibited Wnt/ß-catenin signaling in colon cancer cell lines, likely through TNKS inhibition. Consistent with this possibility, E7449 stabilized axin and TNKS proteins resulting in ß-catenin de-stabilization and significantly altered expression of Wnt target genes. Notably, hair growth mediated by Wnt signaling was inhibited by E7449. A pharmacodynamic effect of E7449 on Wnt target genes was observed in tumors, although E7449 lacked single agent antitumor activity in vivo, a finding typical for selective TNKS inhibitors. E7449 antitumor activity was increased through combination with MEK inhibition. Particularly noteworthy was the lack of toxicity, most significantly the lack of intestinal toxicity reported for other TNKS inhibitors. E7449 represents a novel dual PARP1/2 and TNKS1/2 inhibitor which has the advantage of targeting Wnt/ß-catenin signaling addicted tumors. E7449 is currently in early clinical development.

Comparative gene expression profiling in three primary human cell lines after treatment with a novel inhibitor of Rho kinase or atorvastatin.

Inhibitors of Rho kinase (ROCK) are a relatively new class of drugs with potential benefits in oncology, neurology, and fibrotic and cardiovascular diseases. ROCK inhibitors modulate many cellular functions, some of which are similar to the pleiotropic effects of statins, suggesting additive or synergistic properties. Studies to date have used compounds that inhibit both isoforms of ROCK, ROCK1 and ROCK2. This study was designed to compare gene expression profiles of atorvastatin with the newly developed ROCK2 inhibitor SLx-2119 in primary cultures of normal human endothelial cells, smooth muscle cells, and fibroblasts. Cells were treated with each compound for 24 h, after which total RNA was isolated and genome-wide gene-expression profiles were obtained with Illumina arrays. Because of the known effect of statins on the actin cytoskeleton and on connective tissue growth factor, a prominent growth factor involved in tissue fibrosis, the effects of SLx-2119 and atorvastatin on the actin cytoskeleton and connective tissue growth factor mRNA were also examined in cultures of smooth muscle cells with a fibrotic phenotype, isolated from biopsies of human intestine with radiation-induced fibrosis. Although SLx-2119 and atorvastatin affected expression of genes belonging to the same biological processes, individual genes were mostly different, consistent with synergistic or additive properties. Both SLx-2119 and atorvastatin reduced connective tissue growth factor mRNA and remodeled the actin cytoskeleton in fibrosis-derived smooth muscle cells, suggesting that both compounds have antifibrotic properties. These results form the basis for further studies to assess the possible therapeutic benefit of combined treatments.

In vitro assay of angiogenesis: inhibition of capillary tube formation.

The growth of new blood vessels, or angiogenesis, is a naturally occurring process in both health and disease states. An area of active research, regulation of angiogenesis, is being studied as an approach for the treatment of cancer and a range of other disorders having vascular proliferation as a component. The process of angiogenesis is very complex and occurs in multiple steps, with a major involvement of endothelial cells. Various in vivo models have been developed to assess inhibitors of angiogenesis. As these are generally technically difficult and labor intensive, with observed effects difficult to quantify, they do not lend themselves to compound screening. Rather they are used for confirmatory studies. In contrast, in vitro assays developed to model various steps in the angiogenesis process are easy to perform and lend themselves to high-throughput analysis. Described in this unit is an in vitro assay that can be employed to investigate endothelial differentiation inhibitors through assessment of their effects on capillary tube formation by endothelial cells on Matrigel.

Telehealth videoconferencing: improving home parenteral nutrition patient care to rural areas of Ontario, Canada.

BACKGROUND: Telehealth videoconferencing is a medium for health care professionals to communicate and care for patients living in remote areas. The aim of this study was to provide a survey to examine management outcome of home parenteral nutrition (HPN) patients when followed by telehealth as an alternative modality of care. METHODS: Twenty-six individuals who were identified to benefit from tele-health were invited to participate in a satisfaction survey. The survey was sent to patients by postal mail. The survey also documented the incidence of line sepsis and other medical HPN complications. A cost analysis was also performed according to technology, human resources, and infrastructure. RESULTS: Eighty-one telehealth videoconference sessions have been held since the inception of telehealth in 2002. Of the current telehealth patients, 13 were eligible for the survey. The satisfaction survey response rate was 11/13 (84.6%). The average line sepsis rate for the 13 patients was 0.89/1000 catheter-days. All patients were generally satisfied with videoconferencing as an alternative method of communication and care for new consultation, patient and family education, and follow-up. Travel time and costs to the patients, their families, and the health care system were significantly less. For example, a patient who resides 611 km from Toronto would cost CDN (Canadian) 724.00 dollars for flight and accommodation to meet with the team at the HPN clinic in Toronto. CONCLUSION: Telehealth incorporated the cost-saving ability for HPN patients to maintain proper medical care, support, and collaboration of specialists inaccessible to their local community. Thus, its strongly positive role in HPN care deserves further consideration for a national application.

E-Clinic: an innovative approach to complex symptom management for allogeneic blood and stem cell transplant patients.

The allogeneic blood and stem cell program (ABSCP) at Princess Margaret Hospital, Toronto, performs 75 transplants annually. Many patients live greater than 100 kilometres from the centre and require frequent visits to the hospital for posttransplant care. The weekly travel to clinic, combined with complex symptom issues and the overwhelming desire to be cared for in their home community, is a major burden to patients and care providers. Our team of oncology health professionals, led by the nurse practitioner on service, sought to determine whether telehealth videoconferencing would be a viable option as a care delivery model to meet the complex needs of our remote patients and care partners. We introduced telehealth into the ambulatory clinic as a pilot project in early 2005. Patients were selected based upon symptoms, therapeutic plan and geographical remoteness. Patient progress was monitored with a goal of transitioning patients from posttransplant hospital-based care to partnered self-care in their home communities. The purpose of this article is to illustrate the ABSCP telehealth program development using a patient case study, and to detail the clinical process improvements and overall program successes that have led to the integration of telehealth into everyday clinical practice as a viable service delivery option for patient-centred symptom management and treatment compliance with a geographically remote patient population.

14-3-3 proteins in Schistosoma mansoni; identification of a second epsilon isoform.

A new member of the 14-3-3 protein family in Schistosoma mansoni has been identified. Sequence analysis demonstrated that this protein is a member of the epsilon sub-group and is the orthologue of Schistosoma japonicum 14-3-3epsilon. Since we had previously identified a 14-3-3epsilon protein from S. mansoni, we termed the original protein 14-3-3epsilon-1 and this second epsilon protein 14-3-3epsilon-2. Schistosoma mansoni encodes at least four different 14-3-3 isoforms: the two epsilon proteins and 14-3-3 protein 1 and protein 2, which are zeta-like isoforms. Phylogenetic analysis demonstrated the early divergence of the epsilon isoforms, and that schistosome proteins 1 and 2 are among the oldest non-epsilon 14-3-3 proteins yet identified. Schistosoma mansoni 14-3-3epsilon-1, 14-3-3epsilon-2, and protein 1 are stage specifically expressed in a similar manner, being absent in cercariae and schistosomula, and abundant in lung stage and adult male and female worms. Protein 2 transcript was not detected at any of the life cycle stages examined. All three detected 14-3-3 isoforms elicit an immune response during infection, with the greatest response directed against protein 1. Binding studies with S. mansoni receptor kinase-1 (SmRK1) and human Raf kinase revealed that the three 14-3-3epsilon isoforms exhibit a preference for target protein binding. Although all three isoforms do bind to both targets, 14-3-3 protein 1 interacts most strongly with Raf, whereas the 14-3-3-1 isoform binds SmRK1 preferentially. These results suggest that the individual 14-3-3 proteins may have evolved to play isoform-specific roles in the development and survival of S. mansoni within its host.

Eukaryotic initiation factor 2 alpha subunit associates with TGF beta receptors and 14-3-3 epsilon and acts as a modulator of the TGF beta response.

Schistosoma mansoni receptor kinase 1 (SmRK1) is a divergent member of the TGF beta receptor family. Intracellular proteins that associate with these receptors are likely to play an important role in signaling. 14-3-3 epsilon is a previously described cytoplasmic protein, which associates with both SmRK1 and the human type I TGF beta receptor (T beta RI); overexpression of 14-3-3 epsilon leads to enhanced TGF beta-mediated signaling by T beta RI. We now describe the identification of S. mansoni eukaryotic translation initiation factor 2 alpha subunit (eIF2 alpha), through its interaction with SmRK1 in a yeast two-hybrid assay. S. mansoni eIF2 alpha also interacts with human TGF beta receptors. Strongest association was demonstrated with kinase inactive receptors, particularly the type II TGF beta receptor (T beta RII). Both T beta RI and T beta RII phosphorylate eIF2 alpha in vitro, at sites other than the previously described eIF2 alpha phosphorylation sites. EIF2 alpha also modulates signaling by TGF beta receptors; however, in contrast to 14-3-3 epsilon, eIF2 alpha overexpression inhibits the TGF beta-driven response. These data suggest a novel function for eIF2 alpha in the TGF beta signaling pathway. In addition, we have demonstrated an independent interaction between eIF2 alpha and 14-3-3 epsilon. Coexpression of 14-3-3 epsilon with eIF2 alpha leads to the abrogation of the inhibitory effect of eIF2 alpha on TGF beta-mediated signaling. The interaction of these two regulatory proteins with each other and with the TGF beta receptors and their relative expression levels are likely to be important in fine-tuning the regulation of TGF beta signal transduction.