Development and Optimization of a Target Engagement Model of Brain IDO Inhibition for Alzheimer's Disease.

BACKGROUND: Indoleamine 2,3-dioxygenase (IDO1) inhibition is a promising target as an Alzheimer's disease (AD) Disease-modifying therapy capable of downregulating immunopathic neuroinflammatory processes. METHODS: To aid in the development of IDO inhibitors as potential AD therapeutics, we optimized a lipopolysaccharide (LPS) based mouse model of brain IDO1 inhibition by examining the dosedependent and time-course of the brain kynurenine:tryptophan (K:T) ratio to LPS via intraperitoneal dosing. RESULTS: We determined the optimal LPS dose to increase IDO1 activity in the brain, and the ideal time point to quantify the brain K:T ratio after LPS administration. We then used a brain penetrant tool compound, EOS200271, to validate the model, determine the optimal dosing profile and found that a complete rescue of the K:T ratio was possible with the tool compound. CONCLUSION: This LPS-based model of IDO1 target engagement is a useful tool that can be used in the development of brain penetrant IDO1 inhibitors for AD. A limitation of the present study is the lack of quantification of potential clinically relevant biomarkers in this model, which could be addressed in future studies.

Alzheimer's disease as an autoimmune disorder of innate immunity endogenously modulated by tryptophan metabolites.

Introduction: Alzheimer's disease (AD) is characterized by neurotoxic immuno-inflammation concomitant with cytotoxic oligomerization of amyloid beta (Aß) and tau, culminating in concurrent, interdependent immunopathic and proteopathic pathogeneses. Methods: We performed a comprehensive series of in silico, in vitro, and in vivo studies explicitly evaluating the atomistic-molecular mechanisms of cytokine-mediated and Aß-mediated neurotoxicities in AD.  Next, 471 new chemical entities were designed and synthesized to probe the pathways identified by these molecular mechanism studies and to provide prototypic starting points in the development of small-molecule therapeutics for AD. Results: In response to various stimuli (e.g., infection, trauma, ischemia, air pollution, depression), Aß is released as an early responder immunopeptide triggering an innate immunity cascade in which Aß exhibits both immunomodulatory and antimicrobial properties (whether bacteria are present, or not), resulting in a misdirected attack upon "self" neurons, arising from analogous electronegative surface topologies between neurons and bacteria, and rendering them similarly susceptible to membrane-penetrating attack by antimicrobial peptides (AMPs) such as Aß. After this self-attack, the resulting necrotic (but not apoptotic) neuronal breakdown products diffuse to adjacent neurons eliciting further release of Aß, leading to a chronic self-perpetuating autoimmune cycle.  AD thus emerges as a brain-centric autoimmune disorder of innate immunity. Based upon the hypothesis that autoimmune processes are susceptible to endogenous regulatory processes, a subsequent comprehensive screening program of 1137 small molecules normally present in human brain identified tryptophan metabolism as a regulator of brain innate immunity and a source of potential endogenous anti-AD molecules capable of chemical modification into multi-site therapeutic modulators targeting AD's complex immunopathic-proteopathic pathogenesis. Discussion:  Conceptualizing AD as an autoimmune disease, identifying endogenous regulators of this autoimmunity, and designing small molecule drug-like analogues of these endogenous regulators represents a novel therapeutic approach for AD.

A Series of 2-((1-Phenyl-1H-imidazol-5-yl)methyl)-1H-indoles as Indoleamine 2,3-Dioxygenase 1 (IDO1) Inhibitors.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a promising therapeutic target in cancer immunotherapy and neurological disease. Thus, searching for highly active inhibitors for use in human cancers is now a focus of widespread research and development efforts. In this study, we report the structure-based design of 2-(5-imidazolyl)indole derivatives, a series of novel IDO1 inhibitors which have been designed and synthesized based on our previous study using N1-substituted 5-indoleimidazoles. Among these, we have identified one with a strong IDO1 inhibitory activity (IC50 =0.16 µM, EC50 =0.3 µM). Structural-activity relationship (SAR) and computational docking simulations suggest that a hydroxyl group favorably interacts with a proximal Ser167 residue in Pocket A, improving IDO1 inhibitory potency. The brain penetrance of potent compounds was estimated by calculation of the Blood Brain Barrier (BBB) Score and Brain Exposure Efficiency (BEE) Score. Many compounds had favorable scores and the two most promising compounds were advanced to a pharmacokinetic study which demonstrated that both compounds were brain penetrant. We have thus discovered a flexible scaffold for brain penetrant IDO1 inhibitors, exemplified by several potent, brain penetrant, agents. With this promising scaffold, we provide herein a basis for further development of brain penetrant IDO1 inhibitors.

Effects of the Novel IDO Inhibitor DWG-1036 on the Behavior of Male and Female 3xTg-AD Mice.

The kynurenine pathway metabolizes tryptophan into nicotinamide adenine dinucleotide, producing a number of intermediary metabolites, including 3-hydroxy kynurenine and quinolinic acid, which are involved in the neurodegenerative mechanisms that underlie Alzheimer's disease (AD). Indolamine 2,3-dioxygenase (IDO), the first and rate-limiting enzyme of this pathway, is increased in AD, and it has been hypothesized that blocking this enzyme may slow the progression of AD. In this study, we treated male and female 3xTg-AD and wild-type mice with the novel IDO inhibitor DWG-1036 (80 mg/kg) or vehicle (distilled water) from 2 to 6 months of age and then tested them in a battery of behavioral tests that measured spatial learning and memory (Barnes maze), working memory (trace fear conditioning), motor coordination and learning (rotarod), anxiety (elevated plus maze), and depression (tail suspension test). The 3xTg-AD mice treated with DWG-1036 showed better memory in the trace fear conditioning task and significant improvements in learning but poorer spatial memory in the Barnes maze. DWG-1036 treatment also ameliorated the behaviors associated with increased anxiety in the elevated plus maze and depression-like behaviors in the tail suspension test in 3xTg-AD mice. However, the effects of DWG-1036 treatment on the behavioral tasks were variable, and sex differences were apparent. In addition, high doses of DWG-1036 resulted in reduced body weight, particularly in females. Taken together, our results suggest that the kynurenine pathway is a promising target for treating AD, but more work is needed to determine the effective compounds, examine sex differences, and understand the side effects of the compounds.

Identification of Potent Indoleamine 2,3-Dioxygenase 1 (IDO1) Inhibitors Based on a Phenylimidazole Scaffold.

Inhibition of indoleamine 2,3-dioxygenase (IDO1) is an attractive immunotherapeutic approach for the treatment of a variety of cancers. Dysregulation of this enzyme has also been implicated in other disorders including Alzheimer's disease and arthritis. Herein, we report the structure-based design of two related series of molecules: N1-substituted 5-indoleimidazoles and N1-substituted 5-phenylimidazoles. The latter (and more potent) series was accessed through an unexpected rearrangement of an imine intermediate during a Van Leusen imidazole synthesis reaction. Evidence for the binding modes for both inhibitor series is supported by computational and structure-activity relationship studies.

Susceptibility to hippocampal kindling seizures is increased in aging C57 black mice.

The incidence of seizures increases with old age. Stroke, dementia and brain tumors are recognized risk factors for new-onset seizures in the aging populations and the incidence of these conditions also increased with age. Whether aging is associated with higher seizure susceptibility in the absence of the above pathologies remains unclear. We used classic kindling to explore this issue as the kindling model is highly reproducible and allows close monitoring of electrographic and motor seizure activities in individual animals. We kindled male young and aging mice (C57BL/6 strain, 2-3 and 18-22 months of age) via daily hippocampal CA3 stimulation and monitored seizure activity via video and electroencephalographic recordings. The aging mice needed fewer stimuli to evoke stage-5 motor seizures and exhibited longer hippocampal afterdischarges and more frequent hippocampal spikes relative to the young mice, but afterdischarge thresholds and cumulative afterdischarge durations to stage 5 motor seizures were not different between the two age groups. While hippocampal injury and structural alterations at cellular and micro-circuitry levels remain to be examined in the kindled mice, our present observations suggest that susceptibility to hippocampal CA3 kindling seizures is increased with aging in male C57 black mice.

Risk of Spermatic Cord Injury During Anterior Pelvic Ring and Acetabular Surgery: An Anatomical Study.

BACKGROUND: Anterior pelvic ring surgery includes a variety of plating techniques and insertion of retrograde superior pubic ramus screws. Anterior acetabular surgery also includes fixation through an ilioinguinal or Stoppa approach. These exposures risk injury to the spermatic cord and accompanying genital branch of the genitofemoral nerve. The primary aim of this study was to identify the distance between the midline and the spermatic cords in adult male cadaveric specimens. The secondary aim was to determine spermatic cord diameters and measure the distance between the spermatic cord and implant during instrumentation of a retrograde superior pubic ramus medullary screw. METHODS: Extended Pfannenstiel and Stoppa approaches were performed on 18 embalmed male cadavers bilaterally. Spermatic cord characteristics were recorded and a number of measurements were performed to determine the distance of implants and the midline from the spermatic cord. RESULTS: The average distance between the midline and spermatic cords was 34.2 mm. The average distance between the spermatic cord and implant was 18.2 mm. Eleven of the thirty-six dissections had abnormalities including cord lipomas and inguinal hernias. The average cord diameter was 18.6 mm. The average cord diameter in those with abnormalities was 24.9 mm and 16 mm in those without abnormalities, this difference was statistically significant. DISCUSSION: Due to the proximity of the spermatic cord, the surgeon should either formally expose the cord or limit lateral dissection from the midline during Pfannenstiel and Stoppa exposures. Similarly, the surgeon should use soft-tissue sleeves and oscillating drills to avoid injury to the contralateral spermatic cord during the insertion of retrograde superior pubic ramus medullary screws.

Inguinal Abnormalities in Male Patients with Acetabular Fractures Treated Using an Ilioinguinal Exposure.

BACKGROUND: Surgeons performing an ilioinguinal exposure for acetabular fracture surgery need to be aware of aberrant findings such as inguinal hernias and spermatic cord lesions. The purpose of this study is to report these occurrences in a clinical series of adult males undergoing acetabular fracture fixation and a series of adult male cadavers. The secondary aim is to characterize these abnormalities to aid surgeons in detecting these abnormalities preoperatively and coordinating a surgical plan with a general surgeon. METHODS: Clinical study- Retrospective review of treated acetabular fractures through an ilioinguinal approach. Incidence of inguinal canal and spermatic cord abnormalities requiring general surgery consultation were identified. Corresponding CT scans were reviewed and radiographic characteristics of the spermatic cord abnormalities and/or hernias were noted. Cadaveric study- 18 male cadavers dissected bilaterally using an ilioinguinal exposure. The inguinal canal and the contents of the spermatic cord were identified and characterized. RESULTS: Clinical Study- 5.7% (5/87) of patients had spermatic cord lesion and/or inguinal hernia requiring general surgical intervention. Preoperative pelvic CT scan review identified abnormalities noted intraoperatively in four of the five patients. Cord lipomas visualized as enlargements of the spermatic cord with homogeneous density. Hernias visualized as enlarged spermatic cords with heterogeneous density. Cadaver Study- 31% (11/36) of cadavers studied had spermatic cord and/or inguinal canal abnormalities. Average cord diameter in those with abnormalities was 24.9 mm (15-28) compared to 16 mm (11-22) in normal cords, which was statistically significant. DISCUSSION: The clinical and cadaveric findings emphasize the importance of understanding inguinal abnormalities and the value of detecting them preoperatively. The preoperative pelvic CT scans were highly sensitive in detecting inguinal abnormalities.

A retinoblastoma allele that is mutated at its common E2F interaction site inhibits cell proliferation in gene-targeted mice.

The retinoblastoma protein (pRB) is best known for regulating cell proliferation through E2F transcription factors. In this report, we investigate the properties of a targeted mutation that disrupts pRB interactions with the transactivation domain of E2Fs. Mice that carry this mutation endogenously (Rb1(ΔG)) are defective for pRB-dependent repression of E2F target genes. Except for an accelerated entry into S phase in response to serum stimulation, cell cycle regulation in Rb1(ΔG/ΔG) mouse embryonic fibroblasts (MEFs) strongly resembles that of the wild type. In a serum deprivation-induced cell cycle exit, Rb1(ΔG/ΔG) MEFs display a magnitude of E2F target gene derepression similar to that of Rb1(-/-) cells, even though Rb1(ΔG/ΔG) cells exit the cell cycle normally. Interestingly, cell cycle arrest in Rb1(ΔG/ΔG) MEFs is responsive to p16 expression and gamma irradiation, indicating that alternate mechanisms can be activated in G1 to arrest proliferation. Some Rb1(ΔG/ΔG) mice die neonatally with a muscle degeneration phenotype, while the others live a normal life span with no evidence of spontaneous tumor formation. Most tissues appear histologically normal while being accompanied by derepression of pRB-regulated E2F targets. This suggests that non-E2F-, pRB-dependent pathways may have a more relevant role in proliferative control than previously identified.

Reamer-irrigator-aspirator bone graft and bi Masquelet technique for segmental bone defect nonunions: a review of 25 cases.

INTRODUCTION: Segmental bone loss, either from trauma, tumor or infection is a challenging clinical entity. Amputation is a possible outcome and part of the decision making process. Surgical management is almost always needed and can require several interventions to obtain bone union. A staged protocol of obtaining a clean viable soft tissue bed, placement of a PMMA antibiotic impregnated spacer to induce a neovascular and bioactive membrane followed by autogenous bone graft has been reported with good outcomes. Our study attempts to expand on this data by evaluating the use of RIA bone graft for the treatment of segmental bone loss nonunions following trauma and or infection. METHODS: Following IRB approval, two orthopaedic trauma fellowship trained surgeons used one surgical protocol for the management of segmental bone defect nonunions. Femur RIA bone graft was used as the graft source when possible. We retrospectively evaluated patients with segmental bone loss of the lower extremity over a two year period. Our primary endpoint was clinical and radiographic bone union. A secondary endpoint was RIA related complications. Additionally, by using some known mathematical equations, we show a plausible way of quantifying the amount of bone loss from a long bone based on the shape of the bone, defect shape and the measured length of bone loss on plain radiograph. RESULTS: 25 patients with 27 segmental bone loss nonunions were evaluated. Nineteen were tibia bone loss and eight were femoral. 15 (56%) nonunions were open fractures with bone loss and 12(46%) were for bone loss related to infection or surgical debridement. The average deficit size was 5.8 cm in length (range 1-25 cm). At six months and 1 year post operative, 70% and 90% nonunions were healed clinically and radiographically respectively. There were no RIA related complications. DISCUSSION: RIA bone graft has been shown to be a very bioactive material. Several studies support the use of this bone graft for the treatment of nonunion including one recent study evaluating 13 patients with segmental bone loss. Our study expands on this data by evaluating its use as the primary source of bone graft for the treatment of segmental bone loss nonunions in the lower extremity. CONCLUSION: RIA bone graft for the treatment of segmental bone defect nonunion of the lower extremity appears safe and can yield predictable results when following sound surgical principles. 90% of our nonunions were healed at one year following a single bone graft procedure. Very large defects, once a formidable clinical dilemma can be managed successfully with the use of RIA bone graft.

Ultra-rapid categorization requires visual attention: Scenes with multiple foreground objects.

Human observers can determine whether natural scenes contain an animal or not on the basis of as little as 20 ms viewing; a phenomenon termed ultra-rapid categorization (URC). Recent studies have suggested that URC is unimpaired even when attention resources are concurrently devoted to a second task. This apparent independence of URC from availability of attention resources presents a challenge for the conventional view of high-level vision as attention-demanding. However, one notable feature of the scenes employed in those experiments is that they almost universally comprised only one or two foreground objects. Here, we investigate whether these findings generalize to more complex scenes, more typical of those in nature. We find that categorization of scenes with four primary foreground objects is greatly impaired when attention resources are limited under dual-task conditions, even when scenes are presented for 500 ms. In contrast, URC of scenes with one foreground object is only mildly impaired-the magnitude of this impairment being equivalent to that observed for single objects presented in isolation without naturalistic scene backgrounds. We conclude that URC of complex scenes is particularly attention-dependent but that some attention resources are probably necessary even for URC of simple one-object scenes.

The effect of resident work hour regulations on orthopaedic surgical education.

Accreditation Council for Graduate Medical Education (ACGME) resident work hour regulations have been effective since July 2003. Several areas affected by these changes have been identified, including surgical education. In the current study, the authors evaluated the impact of these changes on surgical education at a two-person-per-year orthopaedic training program. Operative case experiences of PGY 2 and 3 residents during the academic years 2002-2003 and 2003-2004 were compared utilizing ACGME case logs. A data entry log was also distributed to examine subjectively the effects on operative case load. ACGME data showed that PGY 2 and 3 residents performed 21.5% fewer cases between years. The average number of cases per rotation decreased by 20.44% (p =.009, paired t-test). Subjective results also showed a decrease, with an average of 10.8% of cases missed per resident. This study shows that residents who have begun training post-80-hour work week will do fewer procedures. This may result in a decreased level of skill, or it may shift operative experience to the senior resident years, prolonging the learning curve. Regardless, future analysis must be done to determine the full impact on training of the orthopaedic resident.