Modulation of Isomerization and Ligand Exchange Rates by π Bonding in Bis(iminoxolene)iridium Pyridine Complexes.

The bis(iminoxolene)iridium complex (Diso)2IrCl (Diso = N-(2,6-diisopropylphenyl)-4,6-di-tert-butyl-2-imino-o-benzoquinone) reacts with pyridine to give trans-(Diso)2Ir(py)Cl as the kinetic product, with cis-(Diso)2Ir(py)Cl formed as the exclusive thermodynamic product upon heating. Electronic spectra and density functional theory calculations point to very similar electronic structures for the cis and trans isomers, with a nonbonding iminoxolene-centered HOMO and a metal-iminoxolene π* LUMO. The triplet states of cis-(Diso)2Ir(py)Cl and cis-[(Diso)2Ir(py)2]+ (but not trans-(Diso)2Ir(py)Cl) are unusually low in energy (1000-1500 cm-1 above the singlets), as shown by variable-temperature NMR spectroscopy. The low-energy triplets are attributed to a change in dihedral angle in the iminoxolenes, which allows a partial π interaction that cannot be achieved in the trans octahedral compounds. Mechanistic studies of the trans-cis isomerization in toluene indicate that the reaction proceeds via isomerization of the five-coordinate species to a form with cis iminoxolene ligands and an apical oxygen. This form is high in energy due to the loss of a secondary iminoxolene-to-iridium π-donor interaction that is possible in the trans form but not in the cis form for the square pyramidal structures. This stereoelectronic effect, combined with the poorer binding of pyridine in trans-(Diso)2Ir(py)Cl due to the interactions of the N-aryl substituents with the pyridine, makes the pyridine dissociate faster from the trans isomer by a factor of 108 at room temperature.

Preclinical efficacy studies in investigator brochures: Do they enable risk-benefit assessment?

Human protection policies require favorable risk-benefit judgments prior to launch of clinical trials. For phase I and II trials, evidence for such judgment often stems from preclinical efficacy studies (PCESs). We undertook a systematic investigation of application materials (investigator brochures [IBs]) presented for ethics review for phase I and II trials to assess the content and properties of PCESs contained in them. Using a sample of 109 IBs most recently approved at 3 institutional review boards based at German Medical Faculties between the years 2010-2016, we identified 708 unique PCESs. We then rated all identified PCESs for their reporting on study elements that help to address validity threats, whether they referenced published reports, and the direction of their results. Altogether, the 109 IBs reported on 708 PCESs. Less than 5% of all PCESs described elements essential for reducing validity threats such as randomization, sample size calculation, and blinded outcome assessment. For most PCESs (89%), no reference to a published report was provided. Only 6% of all PCESs reported an outcome demonstrating no effect. For the majority of IBs (82%), all PCESs were described as reporting positive findings. Our results show that most IBs for phase I/II studies did not allow evaluators to systematically appraise the strength of the supporting preclinical findings. The very rare reporting of PCESs that demonstrated no effect raises concerns about potential design or reporting biases. Poor PCES design and reporting thwart risk-benefit evaluation during ethical review of phase I/II studies.

High Constitutive Interleukin 10 Level Interferes With the Immune Response to Varicella-Zoster Virus in Elderly Recipients of Live Attenuated Zoster Vaccine.

INTRODUCTION: Live attenuated zoster vaccine (Zostavax) was used to test the hypothesis that constitutive level of interleukin 10 (IL-10), which may be high in elderly subjects, impairs vaccine efficacy. If constitutive IL-10 impairs vaccine efficacy, the effectiveness of viral vaccines might be improved by transient inhibition of IL-10 before vaccination. METHODS: Zostavax was given to 26 patients (age, 60-80 years). IL-10 and immunity to varicella zoster virus (VZV) were measured at baseline and after vaccination. Fluorescent antibody to membrane antigen (FAMA) assays and glycoprotein enzyme-linked immunosorbent assays (gpELISAs) were used to assess humoral immunity; anti-varicella virus T-cell responses were studied in a subset of subjects. In a prospective animal model, T-cell responses to chimeric vaccines against lymphocytic choriomeningitis virus (LCMV) were assessed in mice that express or lack IL-10. RESULTS: FAMA assays revealed significant boosting (by 4-fold) of humoral immunity, which occurred only in subjects (10 of 26) with a low constitutive IL-10 level (ie, <20 pg/mL); moreover, the Zostavax-induced FAMA and gpELISA responses were inversely related to the constitutive IL-10 level. Significant VZV-specific T-cell responses followed vaccination only in subjects with a low constitutive IL-10 level. Vaccine-induced LCMV-specific T-cell responses in mice lacking IL-10 were greater than in wild-type animals. CONCLUSIONS: A high constitutive IL-10 level adversely affects vaccine efficacy.

Ethics Reporting in Biospecimen and Genetic Research: Current Practice and Suggestions for Changes.

Modern approaches for research with human biospecimens employ a variety of substantially different types of ethics approval and informed consent. In most cases, standard ethics reporting such as "consent and approval was obtained" is no longer meaningful. A structured analysis of 120 biospecimen studies recently published in top journals revealed that more than 85% reported on consent and approval, but in more than 90% of cases, this reporting was insufficient and thus potentially misleading. Editorial policies, reporting guidelines, and material transfer agreements should include recommendations for meaningful ethics reporting in biospecimen research. Meaningful ethics reporting is possible without higher word counts and could support public trust as well as networked research.

Moving toward a paradigm shift in the regulatory requirements for pediatric medicines.

Over the past two decades, there has been growing concern over the lack of proper medication for children. This review attempts to evaluate the current progress of EU Pediatric Regulation made since 2007. The lack of properly evaluated pediatric medication has for long been a source of concern in the European Union. The drugs that were used in the past were often not properly evaluated, and dosage was arbitrarily calculated. Therefore, it was necessary to establish the Pediatric Regulation (EC no. 1901/2006) in the EU which would mandate research for pediatric drugs. Current legislations in place not only require mandatory research by pharma industry but also have guidelines to direct the quality of pediatric research performed. The main aim of this regulation was to advance high-quality research and development of pediatric drugs, thereby increasing the availability of safe and effective drugs for children. It also aimed to improve the information available on existing pediatric drugs. It has been 9 years since the pediatric regulation was framed. The pharma industry now sees pediatric research as an integral process of development. Drug companies which develop plans for a new drug, new form of drug, new indication, or new route of administration for adults are obliged to integrate in their development plan similar research for pediatric populations as well. CONCLUSION: It is hoped that the implementation of the current legislation will be reflected better in the future by the marketing of better and safer drugs for the pediatric population. The upcoming assessment to the European Commission in 2017 will further inform us on the impact after 10 years implementation of the legislation. What is Known: • The lack of properly evaluated pediatric medication has for long been a source of concern in the European Union. • Therefore, it was necessary to establish the EU Pediatric Regulation which would mandate research for pediatric drugs. What is New: • It has been 9 years since the pediatric regulation was framed, and the teething problems are slowly being overcome and the regulation is being used with increasing confidence. • As the Regulation is due for revision in 2017, this paper gives a current perspective on the impact of the regulation on availability and access to medicine for children.

Fidelity of implementation to a care team redesign and improved outcomes of diabetes care.

OBJECTIVE: We assessed fidelity of implementation (FOI) to the intended features of a primary care team redesign that integrated registered nurse care managers and patient health coaches onto existing care teams. The relation of FOI ranking and improvements in intermediate outcomes of diabetes care was examined. DESIGN: We assessed FOI by interviewing frontline primary care team members (n = 20). We explored the relation of FOI and outcomes of diabetes care (n = 10 206 patients) over a 3-year period (2010-12). Multilevel, multivariate regression estimated the relation of FOI and improvements in outcomes of diabetes care. SETTING: Five primary care practices in greater Los Angeles, CA, USA. PARTICIPANTS: Ten thousand, two hundred and six adult patients with diabetes; 20 frontline primary care clinicians and staff. MAIN OUTCOME MEASURES: Blood pressure, hemoglobin A1c (HbA1c) and low-density lipoprotein cholesterol (LDL-C) control among adult diabetic patients. RESULTS: All practices improved diabetic patients' LDL-C control over time. In adjusted analyses, the practice with the highest FOI achieved the largest improvement in blood pressure and HbA1c control among diabetic patients. In contrast, the practice with the lowest FOI had the least improvements in blood pressure, HbA1c and LDL-C control. FOI was an inconsistent predictor of intermediate outcomes of diabetes care for other practices. CONCLUSIONS: FOI assessment can be useful for identifying low FOI to a redesign so that technical assistance and resources can be provided to improve team functioning and patient outcomes. High FOI can enable greater improvements in patient outcomes in the context of primary care practice redesign.

Differential skin test reactivity to pollens in pollen food allergy syndrome versus allergic rhinitis.

BACKGROUND: Pollen food allergy syndrome (PFAS), also called oral allergy syndrome, is a form of food allergy in which uncooked foods cause allergic symptoms generally limited to the oral mucosa. It occurs in a subset of patients with pollen allergy, although not all patients have prominent rhinitis symptoms. PFAS is related to antigenic similarity between the pollen and food allergen. OBJECTIVE: The size of skin test reactions in a group of subjects with pollen sensitivity with PFAS was compared with a group of subjects who were pollen sensitive and without PFAS. Self-reported rhinitis symptoms between the two groups were compared to identify if symptom severity differed. METHODS: Twenty subjects with PFAS and 20 subjects with seasonal allergic rhinitis without PFAS were enrolled in the study. All the subjects underwent standard skin-prick testing to a panel of common allergens, including select fresh fruits and vegetables. The subjects completed a Mini Rhinoconjunctivitis Quality of Life Questionnaire as part of their clinical evaluation. The subjects with PFAS and those without PFAS were compared statistically. RESULTS: The subjects with PFAS had significantly larger-sized skin-prick test results specific to pollens (p < 0.05). Despite the larger-sized skin-prick test results, the subjects with allergic rhinitis and PFAS reported milder nasal symptoms in relation to pollen skin test result size when compared with allergic rhinitis controls without PFAS. CONCLUSIONS: Our study outlined basic differences between two seemingly similar patient groups with a particularly striking discordance between skin test result sizes and rhinitis symptoms. This discordance should be explored further to increase mechanistic understanding of allergen cross-reactivity in PFAS.

A randomized, double-blind, placebo-controlled study of histone deacetylase type 6 inhibition for the treatment of painful diabetic peripheral neuropathy.

Introduction: Current treatments for painful diabetic peripheral neuropathy (DPN) are insufficiently effective for many individuals and do not treat nonpain signs and symptoms. The enzyme histone deacetylase type 6 (HDAC6) may play a role in the pathophysiology of painful DPN, and inhibition of HDAC6 has been proposed as a potential treatment. Objectives: To assess the efficacy and safety of the novel HDAC6 inhibitor ricolinostat for the treatment of painful diabetic peripheral neuropathy. Methods: We conducted a 12-week randomized, double-blind, placebo-controlled phase 2 study of the efficacy of ricolinostat, a novel selective HDAC6 inhibitor, in 282 individuals with painful DPN. The primary outcome was the change in the patient-reported pain using a daily diary, and a key secondary outcome was severity of nonpain neuropathic signs using the Utah Early Neuropathy Scale (UENS) score. Results: At the 12-week assessment, changes in average daily pain and UENS scores were not different between the ricolinostat and placebo groups. Conclusion: These results do not support the use of the HDAC6 inhibitor ricolinostat as a treatment for neuropathic pain in DPN for periods up to 12 weeks.

Identification and characterization of noncalcemic, tissue-selective, nonsecosteroidal vitamin D receptor modulators.

Vitamin D receptor (VDR) ligands are therapeutic agents for the treatment of psoriasis, osteoporosis, and secondary hyperparathyroidism. VDR ligands also show immense potential as therapeutic agents for autoimmune diseases and cancers of skin, prostate, colon, and breast as well as leukemia. However, the major side effect of VDR ligands that limits their expanded use and clinical development is hypercalcemia that develops as a result of the action of these compounds mainly on intestine. In order to discover VDR ligands with less hypercalcemia liability, we sought to identify tissue-selective VDR modulators (VDRMs) that act as agonists in some cell types and lack activity in others. Here, we describe LY2108491 and LY2109866 as nonsecosteroidal VDRMs that function as potent agonists in keratinocytes, osteoblasts, and peripheral blood mononuclear cells but show poor activity in intestinal cells. Finally, these nonsecosteroidal VDRMs were less calcemic in vivo, and LY2108491 exhibited more than 270-fold improved therapeutic index over the naturally occurring VDR ligand 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in an in vivo preclinical surrogate model of psoriasis.

In-vivo optical detection of cancer using chlorin e6--polyvinylpyrrolidone induced fluorescence imaging and spectroscopy.

BACKGROUND: Photosensitizer based fluorescence imaging and spectroscopy is fast becoming a promising approach for cancer detection. The purpose of this study was to examine the use of the photosensitizer chlorin e6 (Ce6) formulated in polyvinylpyrrolidone (PVP) as a potential exogenous fluorophore for fluorescence imaging and spectroscopic detection of human cancer tissue xenografted in preclinical models as well as in a patient. METHODS: Fluorescence imaging was performed on MGH human bladder tumor xenografted on both the chick chorioallantoic membrane (CAM) and the murine model using a fluorescence endoscopy imaging system. In addition, fiber optic based fluorescence spectroscopy was performed on tumors and various normal organs in the same mice to validate the macroscopic images. In one patient, fluorescence imaging was performed on angiosarcoma lesions and normal skin in conjunction with fluorescence spectroscopy to validate Ce6-PVP induced fluorescence visual assessment of the lesions. RESULTS: Margins of tumor xenografts in the CAM model were clearly outlined under fluorescence imaging. Ce6-PVP-induced fluorescence imaging yielded a specificity of 83% on the CAM model. In mice, fluorescence intensity of Ce6-PVP was higher in bladder tumor compared to adjacent muscle and normal bladder. Clinical results confirmed that fluorescence imaging clearly captured the fluorescence of Ce6-PVP in angiosarcoma lesions and good correlation was found between fluorescence imaging and spectral measurement in the patient. CONCLUSION: Combination of Ce6-PVP induced fluorescence imaging and spectroscopy could allow for optical detection and discrimination between cancer and the surrounding normal tissues. Ce6-PVP seems to be a promising fluorophore for fluorescence diagnosis of cancer.

Molecular profiling of angiogenesis in hypericin mediated photodynamic therapy.

BACKGROUND: Photodynamic therapy (PDT) involves the administration of a tumor-localizing photosensitizing drug, which is activated by light of specific wavelength in the presence of molecular oxygen thus generating reactive oxygen species that is toxic to the tumor cells. PDT selectively destroys photosensitized tissue leading to various cellular and molecular responses. The present study was designed to examine the angiogenic responses at short (0.5 h) and long (6 h) drug light interval (DLI) hypericin-PDT (HY-PDT) treatment at 24 h and 30 days post treatment in a human bladder carcinoma xenograft model. As short DLI targets tumor vasculature and longer DLI induces greater cellular damage, we hypothesized a differential effect of these treatments on the expression of angiogenic factors. RESULTS: Immunohistochemistry (IHC) results showed minimal CD31 stained endothelium at 24 h post short DLI PDT indicating extensive vascular damage. Angiogenic proteins such as vascular endothelial growth factor (VEGF), tumor necrosis growth factor-alpha (TNF-alpha), interferon-alpha (IFN-alpha) and basic fibroblast growth factor (bFGF) were expressed to a greater extent in cellular targeting long DLI PDT compared to vascular mediated short DLI PDT. Gene expression profiling for angiogenesis pathway demonstrated downregulation of adhesion molecules - cadherin 5, collagen alpha 1 and 3 at 24 h post treatment. Hepatocyte growth factor (HGF) and Ephrin-A3 (EFNA3) were upregulated in all treatment groups suggesting a possible activation of c-Met and Ephrin-Eph signaling pathways. CONCLUSION: In conclusion, long DLI HY-PDT induces upregulation of angiogenic proteins. Differential expression of genes involved in the angiogenesis pathway was observed in the various groups treated with HY-PDT.

Improved formulation of photosensitizer chlorin e6 polyvinylpyrrolidone for fluorescence diagnostic imaging and photodynamic therapy of human cancer.

An improved formulation of the photosensitizer chlorin e6 (Ce6) in combination with the hydrophilic polymer polyvinylpyrrolidone (PVP) was investigated for its potential clinical applications in fluorescence diagnosis and photodynamic therapy (PDT) of cancer. This study reports the comparative preclinical biodistribution and efficacy of Ce6 delivered with or without PVP versus dimethyl sulfoxide (DMSO). The safety and fluorescence pharmacokinetics of Ce6-PVP in humans was also accessed. Biodistribution results showed that Ce6-PVP had higher tumor to normal tissue ratio compared to the other formulations. The sensitivity and specificity derived from the area under the receiver operating characteristics curves showed that the formulations were able to discriminate tumor from peritumoral muscle in the following order: Ce6-PVP > Ce6 > Ce6-DMSO. In vitro PDT results showed that Ce6-PVP was found to induce selective phototoxicity in leukemic cells compared to peripheral mononuclear blood cells. In addition, in vivo light irradiation at 1h after Ce6-PVP was found to induce greater tumor necrosis without causing animal toxicity. In patients, preferential accumulation of Ce6-PVP was observed in angiosarcoma lesions compared to normal skin following intravenous administration. In conclusion, PVP significantly enhanced the Ce6 concentration in tumors compared with Ce6 alone and increased the therapeutic index of PDT without any side effects in animal model. No serious adverse events were observed in human as well.

Immune response against angiosarcoma following lower fluence rate clinical photodynamic therapy.

Tumor response to photodynamic therapy (PDT) is dependent on treatment parameters used. In particular, the light fluence rate may be an important determinant of the treatment outcome. In this clinical case report, we describe the response of angiosarcoma to PDT carried out using different fluence rates and drug and light doses. A patient with recurrent multifocal angiosarcoma of the head and neck was recruited for PDT. A new generation chlorin-based photosensitizer, Fotolon, was administered at a dose of 2.0 to 5.7 mg/kg. The lesions were irradiated with 665 nm laser light for a light dose of 65 to 200 J/cm2 delivered at a fluence rate of 80 or 150 mW/cm2. High dose PDT carried out at a high fluence rate resulted in local control of the disease for up to a year; however, the disease recurred and PDT had to be repeated. PDT of new lesions carried out at a lower fluence rate resulted in tumor eradication. More significantly, it also resulted in spontaneous remission of neighboring and distant untreated lesions. Repeat PDT carried out on a recurrent lesion at a lower fluence rate resulted in eradication of both treated and untreated lesions despite the lower total light dose delivered. Immunohistochemical examination of biopsy samples implies that PDT could have activated a cell-mediated immune response against untreated lesions. Subsequent histopathological examination of the lesion sites showed negative for disease. Our clinical observations show that lower fluence rate PDT results in better outcome and also indicate that the fluence rate, rather than the total light dose, is a more crucial determinant of the treatment outcome. Specifically, lower fluence rate PDT appears to activate the body's immune response against untreated lesions.

Preclinical efficacy in therapeutic area guidelines from the U.S. Food and Drug Administration and the European Medicines Agency: a cross-sectional study.

BACKGROUND AND PURPOSE: Therapeutic area guidelines (TAGs) published by the EMA and the FDA offer guidance in planning the launch of a trial in a certain indication. We assessed and compared the guidance on preclinical efficacy of all available TAGs from EMA and FDA. EXPERIMENTAL APPROACH: EMA and FDA websites and databases were searched for all TAGs. A mixed deductive and inductive approach was applied to analyse and cluster content for preclinical efficacy. KEY RESULTS: A total of 114 EMA and 120 FDA TAGs were identified, covering 126 indications. Our core finding is that 75% of EMA TAGs and 58% from the FDA TAGs do not offer any guidance on preclinical efficacy. TAGs varied widely on the extent, nature and detail of guidance. CONCLUSIONS AND IMPLICATIONS: Guidance on preclinical efficacy in a consistent, comprehensive and explicit way that still allows for justified deviations is an important but neglected aspect of transparency for drug development. This transparency would help sponsors in designing preclinical studies and in negotiating more efficiently with regulators.

Assay Guidance Manual: Quantitative Biology and Pharmacology in Preclinical Drug Discovery.

The Assay Guidance Manual (AGM) is an eBook of best practices for the design, development, and implementation of robust assays for early drug discovery. Initiated by pharmaceutical company scientists, the manual provides guidance for designing a "testing funnel" of assays to identify genuine hits using high-throughput screening (HTS) and advancing them through preclinical development. Combined with a workshop/tutorial component, the overall goal of the AGM is to provide a valuable resource for training translational scientists.

Chlorin e6-polyvinylpyrrolidone as a fluorescent marker for fluorescence diagnosis of human bladder cancer implanted on the chick chorioallantoic membrane model.

The use of fluorescence diagnosis as a modern cancer diagnostic modality is rapidly gaining importance in the field of urology. It is based on the detection of distinctive light emission of tissues sensitized by fluorescent dyes, commonly referred to as photosensitizers, after irradiation with a specific light source. Therefore, the search for specific fluorescent dyes with high sensitivity and specificity for bladder cancer is constantly being sought after. The aim of this study is to investigate the use of a new formulation consisting a mixture of chlorin e6 and polyvinylpyrrolidone (Ce6-PVP) for the detection of human bladder cancer cells (MGH) implanted on the chick chorioallantoic membrane (CAM) model. Uptake kinetics studies were quantitatively determined for both systemic and topical administrations of Ce6-PVP to the normal CAM as well as the MGH human bladder tumor implanted on CAM using fluorescence imaging technique. Rapid elimination of Ce6-PVP was displayed following topical application compared to systemic administration in the normal CAM system. Ce6-PVP was found to localize selectively in the xenografted bladder tumor in contrast to the CAM tissue. Neither dark toxicity nor irritancy was observed on the CAM tissue at the dose of 2 mg/kg Ce6-PVP. In conclusion, the Ce6-PVP formulation appeared to have the potential as a fluorescent marker for fluorescence diagnosis of human bladder cancer.

Fluorescence detection of bladder cancer using urine cytology.

Bladder cancer is the fourth most common malignant disease worldwide, accounting for 4% of all cancer cases. In Singapore, it is the ninth most common form of cancer. The high mortality rate in bladder cancer can be reduced by early treatment following pre-cancerous screening. Currently, the gold standard for screening bladder tumors is histological examination of biopsy specimens, which is both invasive and time-consuming. In this study, ex vivo urine fluorescence cytology was investigated to offer an alternative timely and biopsy-free means for detecting bladder cancers. Sediments in patient urine samples were extracted and incubated with a novel photosensitizer, hypericin. Laser confocal microscopy was used to capture the fluorescence images at an excitation wavelength of 488 nm. Images were subsequently processed to single out the exfoliated bladder cancer cells from the other cells based on the cellular size. Intensity histograms of each targeted cell and feature vectors, derived from the histogram moments, were used to represent each sample. A difference in the distribution of the feature vectors of normal and low-grade cancerous bladder cancer cells were observed. A diagnostic algorithm for discriminating between normal and low-grade cancerous cells is elucidated in this report. This study suggests that the fluorescence intensity profiles of hypericin in bladder cells can potentially provide an automated quantitative means of early bladder cancer diagnosis.

Essential role of the homeodomain for pituitary homeobox 1 activation of mouse gonadotropin-releasing hormone receptor gene expression through interactions with c-Jun and DNA.

The gonadotropin-releasing hormone receptor (GnRHR) is expressed primarily in the gonadotropes of the anterior pituitary. Pituitary homeobox 1 (Pitx-1) has been shown to activate pituitary-specific gene expression by direct DNA binding and/or protein-protein interaction with other transcription factors. We hypothesized that Pitx-1 might also dictate tissue-specific expression of the mouse GnRHR (mGnRHR) gene in a similar manner. Pitx-1 activated the mGnRHR gene promoter, and transactivation was localized to sequences between -308 and -264. Pitx-1 bound to this region only with low affinity. This region includes an activating protein 1 (AP-1) site, which was previously shown to be important for mGnRHR gene expression. Further characterization indicated that an intact AP-1 site was required for full Pitx-1 responsiveness. Furthermore, Pitx-1 and AP-1 were synergistic in the activation of the mGnRHR gene promoter. A Pitx-1 homeodomain (HD) point mutation, which eliminated DNA binding ability, caused only a partial reduction of transactivation, whereas deletion of the HD completely prevented transactivation. Pitx-1 interacted directly with c-Jun, and the HD was sufficient for this interaction. While the point mutation in the Pitx-1 HD did not affect interaction with c-Jun, deletion of the HD eliminated the interaction. Taken together, our studies indicate that Pitx-1 can direct transactivation of the mGnRHR gene, in part by DNA binding and in part by an action of Pitx-1 as a cofactor for AP-1, augmenting AP-1 activity through a novel protein-protein interaction between c-Jun and the HD of Pitx-1.