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ATP-dependent P2X3 receptors play a crucial role in the sensitization of nerve fibers and pathological pain pathways. They are also involved in pathways triggering cough and may contribute to the pathophysiology of endometriosis and overactive bladder. However, despite the strong therapeutic rationale for targeting P2X3 receptors, preliminary antagonists have been hampered by off-target effects, including severe taste disturbances associated with blocking the P2X2/3 receptor heterotrimer. Here we present a P2X3 receptor antagonist, eliapixant (BAY 1817080), which is both highly potent and selective for P2X3 over other P2X subtypes in vitro, including P2X2/3. We show that eliapixant reduces inflammatory pain in relevant animal models. We also provide the first in vivo experimental evidence that P2X3 antagonism reduces neurogenic inflammation, a phenomenon hypothesised to contribute to several diseases, including endometriosis. To test whether eliapixant could help treat endometriosis, we confirmed P2X3 expression on nerve fibers innervating human endometriotic lesions. We then demonstrate that eliapixant reduces vaginal hyperalgesia in an animal model of endometriosis-associated dyspareunia, even beyond treatment cessation. Our findings indicate that P2X3 antagonism could alleviate pain, including non-menstrual pelvic pain, and modify the underlying disease pathophysiology in women with endometriosis. Eliapixant is currently under clinical development for the treatment of disorders associated with hypersensitive nerve fibers.

Purpose: Post-operative radiotherapy (PORT) for resected cutaneous squamous cell carcinoma (CSCC) with perineural invasion (PNI) is controversial. Therefore, we conducted a survey to review treatment recommendations among Radiation Oncologists (ROs) in the management of CSCC with PNI. Materials & Methods: In March 2011, we contacted all ROs and trainees in the US through email addresses listed in the 2009 ASTRO directory. Our web-based survey presented clinical vignettes involving Mohs micrographically resected CSCC with microscopic PNI (mPNI) or clinical PNI (cPNI). For each vignette, ROs were asked to indicate if PORT was appropriate and to further specify the dose and volume to treat. Results: Three hundred fifty two responses were completed and analyzed. The majority of ROs (72%) had over 10 years of post residency experience. 64% of the sampled ROs had a special interest in treating head and neck cancers, and 64% treated 4 or more cases per year. Approximately 95% recommended PORT for cPNI whereas 59% recommended PORT for mPNI. Post residency experience (10+ yrs vs. = 0.005) and for mPNI of deep subcutaneous non-named nerve involvement (80% vs. 60%, p = 0.001). ROs treating 8 or more cases per year (vs. = 0.01). Conclusions: Our study demonstrates significant variability among ROs in the management of CSCC with mPNI. For cases of cPNI, an overwhelming majority recommended PORT. In cases of mPNI, there was no consensus for recommending PORT, although experienced practitioners had a lower threshold for offering treatment. These results indicate the need for prospective clinical studies to clarify the role of PORT in CSCC patients with mPNI.

Objective To evaluate whether Mohs surgeons' management of high-risk cutaneous squamous cell carcinoma (HRCSCC) is uniform regarding radiologic nodal staging (RNS) and adjuvant radiation therapy (ART). Design A survey study of randomly selected, fellowship-trained Mohs surgeons. Setting An academic medical center. Participants American College of Mohs Surgery members who responded to an e-mail invitation completed either a survey regarding management of HRCSCC (n = 117) or SCC with perineural invasion (PNI) (n = 118). Participants totaled approximately 25% of the American College of Mohs Surgery membership. Main Outcome Measures (1) Percentage of patients with HRCSCC referred for RNS, sentinel lymph node biopsy (SLNB), or ART over the preceding 12 months; (2) top factors leading surgeons to consider RNS, SLNB, or ART; and (3) acceptance of ART for clinical scenarios of various degrees of PNI. Results Most respondents cited PNI and in-transit metastasis as top factors leading to consideration of RNS, SLNB, or ART. Otherwise, there was no consensus regarding use of, or indications for, RNS, SLNB, or ART. Conclusions The lack of consistency between experts indicates that there is equipoise regarding indications for RNS and ART in HRCSCC. There is also wide variation in RNS and ART practices among Mohs surgeons who are specifically trained to manage HRCSCC. Clinical trials should therefore be conducted in these areas as there is no clear standard of care.

Cutaneous T-cell lymphomas most commonly have a CD4(+) memory T-cell phenotype with relatively indolent course, but may in rare cases present with a CD8(+) cytotoxic phenotype exhibiting strikingly more aggressive clinical behavior. We present two cases of the clinically aggressive subtype of primary cutaneous epidermotropic CD8(+) cutaneous T-cell lymphoma and review the current literature, clinical behavior, and recommendations for treatment distinct from that of more common CD4(+) variants of cutaneous T-cell lymphoma.

IL-21, a common gamma-chain cytokine secreted by activated CD4+ T cells, influences both humoral and cell-mediated immune responses through the regulation of T, B, dendritic, and natural killer (NK) cells. Sézary syndrome is an advanced form of cutaneous T-cell lymphoma, a clonally derived malignancy of CD4+ T cells that is characterized by profound defects in host cellular immune function. As a modulator of both innate and adaptive immune responses, IL-21 could play an important role in augmenting cell-mediated immunity in these patients. Normal donor and Sézary syndrome patient peripheral blood mononuclear cells were cultured with IL-21 and tested for CD8+ T- and NK-cell activation, NK-cell cytotoxicity, and tumor cell proliferation and apoptosis. IL-21 resulted in a modest increase in CD8+ T- and NK-cell activation, associated with a marked increase in cytolytic activity against both K562 and malignant CD4+ T-cell targets. Although IL-21 failed to demonstrate pro-apoptotic effects on the malignant CD4+ T cells, it is noteworthy that it had no demonstrable proliferative effects on these cells. Thus, IL-21 may play an important role in enhancing the host immune response of Sézary syndrome patients through the increased cytolytic activity of T and NK cells.

Background Because there are currently many effective therapies available for Sezary syndrome, close monitoring of disease progression is required in order for a clinician to know when to institute or change an intervention. It has been our clinical experience that changes in patients' CD4 + CD26 − T-cell populations of peripheral blood lymphocytes herald changes in their clinical status. Objective Our purpose was to evaluate whether a change in patients' CD4 + CD26 − population of T cells presages a change in their clinical status. We also sought to investigate the association between a change in T-cell populations that are CD4 + CD7 − , CD8 + , CD56 + , and the CD4 + /CD8 + T-cell ratio and a change in the patient's clinical status. Methods We conducted a retrospective chart review analysis of 21 patients with Sezary syndrome who had flow cytometry, usually including levels of CD4 + CD26 − , CD4 + CD7 − , CD8 + , CD56 + , and CD4 + /CD8 + ratios measured at two time periods, 12 weeks apart. Results We report two cases in which changes in patients' clinical status were preceded by several weeks by a change in their CD4 + CD26 − level. We report weak associations between a decreasing CD4 + CD26 − T-cell population, a decreasing CD4 + CD7 − population, an increasing CD56 + population, and an improving clinical status. We also report stronger associations between both a decreasing CD8 + population and an increasing CD4 + /CD8 + ratio and a worsening clinical status. Limitations The study was limited by the number of patients and the time period over which the study was conducted. In addition, varying configurations of CD4 + CD26 − T-cell populations were observed that may have limited the utility of this measurement. Conclusions Flow cytometry assays of patients' blood and, in particular, measurement of the CD4 + CD26 − population of lymphocytes over time may be a valuable tool for monitoring patients with Sezary syndrome. There exist varying configurations of CD26 T lymphocytes that may cause differences in standards for what is considered positive and negative between observers. Further prospective analysis involving larger groups of patients is recommended.

Previous studies by others using transplantable murine tumor models have demonstrated that the administration of antibodies that block CTLA-4 interaction with B7 can provoke the elimination of established tumors, and that the tumor suppression is mediated by T-cells and/or cells expressing NK1.1. Studies from our lab have established in a human/severe combined immunodeficient (SCID) mouse chimeric model that autologous peripheral blood leukocytes (PBL) can suppress the growth of tumor xenografts in a PBL dose-dependent fashion, and that this suppression is dependent upon the patient's T and NK cells. Using this human/mouse chimeric model, we sought to determine whether an antibody blockade of CTLA-4 would enhance the anti-tumor response of a patient's PBL. It was first important to determine whether the tumor suppression observed in the SCID model was dependent upon CD28/B7 co-stimulation. Blockade of B7 with a human CTLA-4-Ig fusion protein completely abrogated the lymphocyte-mediated tumor suppression, confirming in this model that tumor suppression is dependent upon a CD28/B7 co-stimulation. Using two different CTLA-4 specific monoclonal antibodies, we observed that CTLA-4 blockade significantly enhanced the human lymphocyte-mediated tumor suppression in mice co-engrafted with PBL and tumor cells. This enhancement was observed in both an allogeneic setting (in which the PBL were allogeneic with respect to the tumor) and an autologous setting (in which the PBL and tumor were from the same patient). These results sustain the notion that human anti-tumor immune response can be augmented (in vivo) by blocking the interaction between CTLA-4 and B7.

Cutaneous T cell lymphomas (CTCLs) are a heterogenous group of lymphoproliferative disorders caused by clonally derived, skin-invasive T cells. Mycosis fungoides (MF) and Sezary syndrome (SS) are the most common types of CTCLs and are characterized by malignant CD4(+)/CLA(+)/CCR4(+) T cells that also lack the usual T cell surface markers CD7 and/or CD26. As MF/SS advances, the clonal dominance of the malignant cells results in the expression of predominantly Th2 cytokines, progressive immune dysregulation in patients, and further tumor cell growth. This review summarizes recent insights into the pathogenesis and immunobiology of MF/SS and how these have shaped current therapeutic approaches, in particular the growing emphasis on enhancement of host antitumor immune responses as the key to successful therapy.

The successful engraftment of human tumors and human immunocompetent cells into severe combined immunodeficient (SCID) mice has led to the generation of a wide array of different experimental designs that have proven useful in studying the cell biology of human cancer, and for evaluating novel therapeutic approaches to the treatment of cancer. In this review five of the most frequently used embodiments of the SCID model are presented. The goals of this review are to discuss how each model has been utilized to study human cancer and its response to many different novel therapies, to provide an assessment of the strengths and limitations of each model, and to outline future directions with a focus on what is needed to overcome some of the current limitations and pitfalls of the SCID models.

Ion channels are a class of proteins that are attractive human, animal, and crop health targets. Ion channels can be considered high-gain (physiologically speaking), high-reward (therapeutic benefit) and high technology (difficult assay development) drug targets. Ion channels are attractive therapeutic targets because they often have high control gain in physiological systems; opening or blocking many of the ion channels studied to date has a large effect on the function of the muscle, nerve, or cell containing the channels. The major issue confronting ion channel drug development teams is safety as the same or closely-related ion channels are often widely distributed in the heart and CNS, and activity at these channels needs to be minimal. This chapter reviews the history of ion channel HTS techniques, using examples from the authors’ work to illustrate how the necessary approaches yield good results, and describing in numerical detail their approach to assay development and full library HTS for selected examples in as much detail as possible.

Genes encoding the p35 and p40 subunits of human interleukin-12 (IL-12) and the bacterial aminoglycoside phosphotransferase were cloned into a mammalian expression plasmid. The resultant plasmid, pCMVIL-12neo, was used to transfect human lung tumor cell lines in vitro. Stably transfected subclones were generated and found to secrete human IL-12 for at least 10 days following a lethal dose of gamma-radiation. The ability of the IL-12--producing tumor cells to promote an antitumor response in vivo was evaluated in SCID mice co-engrafted subcutaneously with human peripheral blood lymphocytes (PBLs) and viable human lung tumor cells (SCID-Winn assay). Using this model system, it was established that IL-12 released locally into tumors by irradiated IL-12--transfected cells activated the human PBL and promoted their ability to suppress tumor development in a dose-dependent fashion. PBL subset depletion studies revealed that the antitumor effect promoted by the IL-12--modified cells was dependent on the presence of human CD8(+) T cells and, to a lesser extent, human CD56(+) natural killer cells within the xenograft. We conclude that (a) irradiated human lung tumor cells genetically modified with pCMVIL-12neo secrete bioactive human IL-12 at concentrations sufficient to promote a human lymphocyte-mediated antitumor response in the microenvironment of the xenograft, and (b) that the SCID-Winn assay provides a useful model for the preclinical evaluation of cytokine-based human immunotherapy protocols.

A new technology for the local and sustained delivery of immunostimulatory molecules to the tumor environment for cancer immunotherapy was evaluated. The ability of cytokines delivered by biodegradable microspheres to promote the antitumor activity of human peripheral blood lymphocytes (PBL) was tested in a human PBL, human tumor, and SCID mouse (SCID-Winn) model. Co-engraftment of human recombinant IL-12-loaded microspheres with human PBL and tumors in SCID mice promoted complete tumor suppression in as many as 100% of the mice, whereas microspheres loaded with polyethyleneglycol-interleukin-2 suppressed but did not eliminate the growth of tumor xenografts. Control microspheres (loaded with bovine serum albumin) in the presence of human PBL or cytokine-loaded microspheres in the absence of human PBL had no tumor-suppressive effect. Coincident with the enhancement of the human PBL-mediated antitumor activity in mice treated with IL-12-loaded microspheres was the production and release of human IFN-gamma indicating that IL-12 released from the microspheres results in the activation of the engrafted human PBL. The results establish that biodegradable microspheres represent an effective tool for the local and sustained delivery of cytokines to the tumor environment for cancer immunotherapy.

Profiling of putative lead compounds against a representative panel of relevant enzymes, receptors, ion channels, and transporters is a pragmatic approach to establish a preliminary view of potential issues that might later hamper development. An early idea of which off-target activities must be minimized can save valuable time and money during the preclinical lead optimization phase if pivotal questions are asked beyond the usual profiling at hERG. The best data for critical evaluation of activity at ion channels is obtained using functional assays, since binding assays cannot detect all interactions and do not provide information on whether the interaction is that of an agonist, antagonist, or allosteric modulator. For ion channels present in human cardiac muscle, depending on the required throughput, manual-, or automated-patch-clamp methodologies can be easily used to evaluate compounds individually to accurately reveal any potential liabilities. The issue of expanding screening capacity against a cardiac panel has recently been addressed by developing a series of robust, high-throughput, cell-based counter-screening assays employing fluorescence-based readouts. Similar assay development approaches can be used to configure panels of efficacy assays that can be used to assess selectivity within a family of related ion channels, such as Nav1.X channels. This overview discusses the benefits of in vitro assays, specific decision points where profiling can be of immediate benefit, and highlights the development and validation of patch-clamp and fluorescence-based profiling assays for ion channels (for examples of fluorescence-based assays, see Bhave et al., 2010; and for high-throughput patch-clamp assays see Mathes, 2006; Schrøder et al., 2008).

Leukemic cell growth in SCID mice has been reported as a predictor of disease relapse. However, there is a paucity of literature regarding xenograft growth and clinical outcomes in non-small cell lung cancer (NSCLC). Seventy-nine specimens from patients with NSCLC were either subcutaneously implanted into SCID mice and/or placed in tissue culture. Retrospective chart review was correlated with stage, histology, necrosis, disease-free interval, and survival. Tumor xenografts were successfully established with 17 of 37 (46%) tumor biopsy tissues. Thirteen of 59 (22%) specimens grew in cell culture. Patients whose tumors grew in SCID mice had no difference in survival compared to those with no growth ( n=20, p=0.42). Median survival was 36 months in 13 patients whose tumors grew in cell culture compared to 39 months in 46 patients without growth. Eight of 12 (67%) patients with metastasis showed SCID/human xenograft growth, whereas nine of 25 (36%) without metastases did so ( p=0.08). Growth of tumor cells in vitro occurred in 11 of 31 (35%) adenocarcinomas, one of 25 (4%) squamous cell carcinomas, and one of three (33%) large cell carcinomas ( p=0.02). Well or moderately differentiated tumors grew in cell culture in only two of 22 (9%), whereas poorly or undifferentiated tumors grew in 11 of 32 (34%) cases ( p=0.03). We conclude that neither the ability of a tumor to engraft and grow in SCID mice nor its ability to grow in vitro in cell culture is a reliable predictor of disease outcome or survival in patients with NSCLC. The ability to propagate tumors in vitro appears to be more dependent upon the histological type of tumor and its degree of differentiation.

The ability to engraft human tumors and human immunocompetent cells successfully in severe combined immunodeficient (SCID) mice has spawned the development and use of human–mouse chimeric models to evaluate anti-cancer therapies. The lack of standardization and many other potential pitfalls have contributed to the current controversy surrounding the reliability of these different models. Five frequently used SCID mouse models and their specific applications are summarized with the specific aim of providing an objective discussion of the strengths and limitations of each model, together with suggestions for overcoming some of the variabilities and for improving the design and use of future models.