Investigating the Utility of Humanized Pregnane X Receptor-Constitutive Androstane Receptor-CYP3A4/7 Mouse Model to Assess CYP3A-Mediated Induction.

Clinical induction liability is assessed with human hepatocytes. However, underpredictions in the magnitude of clinical induction have been reported. Unfortunately, in vivo studies in animals do not provide additional insight because of species differences in drug metabolizing enzymes and their regulatory pathways. To circumvent this limitation, transgenic animals expressing human orthologs were developed. The aim of this work was to investigate the utility of mouse models expressing human orthologs of pregnane X receptor, constitutive androstane receptor, and CYP3A4/7 (Tg-Composite) in evaluating clinical induction. Rifampin, efavirenz, and pioglitazone, which were employed to represent strong, moderate, and weak inducers, were administered at multiple doses to Tg-Composite animals. In vivo CYP3A activity was monitored by measuring changes in the exposure of the CYP3A probe substrate triazolam. After the in vivo studies, microsomes were prepared from their livers to measure changes of in vitro CYP3A4 activity. In both in vivo and in vitro, distinction of clinic induction was recapitulated as rifampin yielded the greatest inductive effect followed by efavirenz and pioglitazone. Interestingly, with rifampin, in vivo CYP3A activity was approximately 4-fold higher than in vitro activity. Conversely, there was no difference between in vivo and in vitro CYP3A activity with efavirenz. These findings are consistent with the report that, although rifampin exhibits differential inductive effects between the intestines and liver, efavirenz does not. These data highlight the promise of transgenic models, such as Tg-Composite, to complement human hepatocytes to enhance the translatability of clinical induction as well as become a powerful tool to further study mechanisms of drug disposition. SIGNIFICANCE STATEMENT: Underprediction of the magnitude of clinical induction when using human hepatocytes has been reported, and transgenic models may improve clinical translatability. The work presented here showcases the human orthologs of pregnane X receptor, constitutive androstane receptor, and CYP3A4/7 model, which was able to recapitulate the magnitude of clinical induction and to differentiate tissue-dependent induction observed with rifampin but not with efavirenz. These results not only foreshadow the potential application of such transgenic models in assessing clinical induction but also in further investigation of the mechanism of drug disposition.

Preclinical Safety Assessment of a Highly Selective and Potent Dual Small-Molecule Inhibitor of CBP/P300 in Rats and Dogs.

Cyclic adenosine monophosphate-response element (CREB)-binding protein (CBP) and EP300E1A-binding protein (p300) are members of the bromodomain and extraterminal motif (BET) family. These highly homologous proteins have a key role in modulating transcription, including altering the status of chromatin or through interactions with or posttranslational modifications of transcription factors. As CBP and p300 have known roles for stimulating c-Myc oncogenic activity, a small-molecule inhibitor, GNE-781, was developed to selectively and potently inhibit the CBP/p300 bromodomains (BRDs). Genetic models have been challenging to develop due to embryonic lethality arising from germline homozygous mutations in either CBP or P300. Hence, the purpose of this study was to characterize the role of dual inhibition of these proteins in adult rats and dogs. Repeat dose toxicity studies were conducted, and toxicologic and pathologic end points were assessed. GNE-781 was generally tolerated; however, marked effects on thrombopoiesis occurred in both species. Evidence of inhibition of erythroid, granulocytic, and lymphoid cell differentiation was also present, as well as deleterious changes in gastrointestinal and reproductive tissues. These findings are consistent with many preclinical (and clinical) effects reported with BET inhibitors targeting BRD proteins; thus, the current study findings indicate a likely important role for CBP/p300 in stem cell differentiation.

Utility of CYP3A4 and PXR-CAR-CYP3A4/3A7 Transgenic Mouse Models To Assess the Magnitude of CYP3A4 Mediated Drug-Drug Interactions.

Species differences in the expression, activity, regulation, and substrate specificity of metabolizing enzymes preclude the use of animal models to predict clinical drug-drug interactions (DDIs). The objective of this work is to determine if the transgenic (Tg) Cyp3a-/-Tg-3A4Hep/Int and Nr1i2/Nr1i3-/--Cyp3a-/-Tg-PXR-CAR-3A4/3A7Hep/Int (PXR-CAR-CYP3A4/3A7) mouse models could be used to predict in vivo DDI of 10 drugs; alprazolam, bosutinib, crizotinib, dasatinib, gefitinib, ibrutinib, regorafenib, sorafenib, triazolam, and vandetinib (as victims); with varying magnitudes of reported CYP3A4 clinical DDI. As an assessment of the effect of CYP3A4 inhibition, these drugs were coadministered to Cyp3a-/-Tg-3A4Hep/Int mice with the CYP3A inhibitor, itraconazole. For crizotinib, regorafenib, sorafenib, and vandetanib, there was no significant increase of AUC observed; with alprazolam, bosutinib, ibrutinib, dasatinib, and triazolam, pretreatment with itraconazole resulted in a 2-, 4-, 17-, 7-, and 15-fold increase in AUC, respectively. With the exception of gefinitib for which the DDI effect was overpredicted (12-fold in Tg-mice vs 2-fold in the clinic), the magnitude of AUC increase observed in this study was consistent (within 2-fold) with the clinical DDI observed following administration with itraconazole/ketoconazole. As an assessment of CYP3A4 induction, following rifampin pretreatment to PXR-CAR-3A4/3A7Hep/Int mice, an 8% decrease in vandetanib mean AUC was observed; 39-52% reduction in AUC were observed for dasatinib, ibrutinib, regorafenib, and sorafenib compared to vehicle treated mice. The greatest effect of rifampin induction was observed with alprazolam, bosutinib, crizotinib, gefitinib, and triazolam where 72-91% decrease in AUC were observed. With the exception of vandetanib for which rifampin induction was under-predicted, the magnitude of induction observed in this study was consistent (within 2-fold) with clinical observations. These data sets suggest that, with two exceptions, these transgenic mice models were able to exclude or capture the magnitude of CYP3A4 clinical inhibition and induction. Data generated in transgenic mice may be used to gain confidence and complement in vitro and in silico methods for assessing DDI potential/liability.

Discovery of TRPA1 Antagonist GDC-6599: Derisking Preclinical Toxicity and Aldehyde Oxidase Metabolism with a Potential First-in-Class Therapy for Respiratory Disease.

Transient receptor potential ankyrin 1 (TRPA1) is a nonselective calcium ion channel highly expressed in the primary sensory neurons, functioning as a polymodal sensor for exogenous and endogenous stimuli, and has been implicated in neuropathic pain and respiratory disease. Herein, we describe the optimization of potent, selective, and orally bioavailable TRPA1 small molecule antagonists with strong in vivo target engagement in rodent models. Several lead molecules in preclinical single- and short-term repeat-dose toxicity studies exhibited profound prolongation of coagulation parameters. Based on a thorough investigative toxicology and clinical pathology analysis, anticoagulation effects in vivo are hypothesized to be manifested by a metabolite─generated by aldehyde oxidase (AO)─possessing a similar pharmacophore to known anticoagulants (i.e., coumarins, indandiones). Further optimization to block AO-mediated metabolism yielded compounds that ameliorated coagulation effects in vivo, resulting in the discovery and advancement of clinical candidate GDC-6599, currently in Phase II clinical trials for respiratory indications.

Tetrahydrofuran-Based Transient Receptor Potential Ankyrin 1 (TRPA1) Antagonists: Ligand-Based Discovery, Activity in a Rodent Asthma Model, and Mechanism-of-Action via Cryogenic Electron Microscopy.

Transient receptor potential ankyrin 1 (TRPA1) is a nonselective calcium-permeable ion channel highly expressed in the primary sensory neurons functioning as a polymodal sensor for exogenous and endogenous stimuli and has generated widespread interest as a target for inhibition due to its implication in neuropathic pain and respiratory disease. Herein, we describe the optimization of a series of potent, selective, and orally bioavailable TRPA1 small molecule antagonists, leading to the discovery of a novel tetrahydrofuran-based linker. Given the balance of physicochemical properties and strong in vivo target engagement in a rat AITC-induced pain assay, compound 20 was progressed into a guinea pig ovalbumin asthma model where it exhibited significant dose-dependent reduction of inflammatory response. Furthermore, the structure of the TRPA1 channel bound to compound 21 was determined via cryogenic electron microscopy to a resolution of 3 Å, revealing the binding site and mechanism of action for this class of antagonists.

A TRPA1 inhibitor suppresses neurogenic inflammation and airway contraction for asthma treatment.

Despite the development of effective therapies, a substantial proportion of asthmatics continue to have uncontrolled symptoms, airflow limitation, and exacerbations. Transient receptor potential cation channel member A1 (TRPA1) agonists are elevated in human asthmatic airways, and in rodents, TRPA1 is involved in the induction of airway inflammation and hyperreactivity. Here, the discovery and early clinical development of GDC-0334, a highly potent, selective, and orally bioavailable TRPA1 antagonist, is described. GDC-0334 inhibited TRPA1 function on airway smooth muscle and sensory neurons, decreasing edema, dermal blood flow (DBF), cough, and allergic airway inflammation in several preclinical species. In a healthy volunteer Phase 1 study, treatment with GDC-0334 reduced TRPA1 agonist-induced DBF, pain, and itch, demonstrating GDC-0334 target engagement in humans. These data provide therapeutic rationale for evaluating TRPA1 inhibition as a clinical therapy for asthma.

Discovery of a Potent (4 R,5 S)-4-Fluoro-5-methylproline Sulfonamide Transient Receptor Potential Ankyrin 1 Antagonist and Its Methylene Phosphate Prodrug Guided by Molecular Modeling.

Transient receptor potential ankyrin 1 (TRPA1) is a non-selective cation channel expressed in sensory neurons where it functions as an irritant sensor for a plethora of electrophilic compounds and is implicated in pain, itch, and respiratory disease. To study its function in various disease contexts, we sought to identify novel, potent, and selective small-molecule TRPA1 antagonists. Herein we describe the evolution of an N-isopropylglycine sulfonamide lead (1) to a novel and potent (4 R,5 S)-4-fluoro-5-methylproline sulfonamide series of inhibitors. Molecular modeling was utilized to derive low-energy three-dimensional conformations to guide ligand design. This effort led to compound 20, which possessed a balanced combination of potency and metabolic stability but poor solubility that ultimately limited in vivo exposure. To improve solubility and in vivo exposure, we developed methylene phosphate prodrug 22, which demonstrated superior oral exposure and robust in vivo target engagement in a rat model of AITC-induced pain.

Magnetic resonance imaging of the collateral ligaments and the anatomic quadrants of the knee.

Injuries to the lateral and medial supporting structures of the knee can be significantly disabling and somewhat difficult to detect and evaluate clinically. An accurate imaging evaluation of these structures requires the use of the appropriate MR imaging sequences and the detailed knowledge of the anatomic structures that are present in these locations. Normal function is dependent on the integrity of the complex functional structures and effective clinical treatment, including surgical repair, of these structures is predicated on an optimal diagnostic evaluation. A successful diagnostic evaluation can expedite treatment and provide the best opportunity for a favorable long-term outcome.

Assessing hERG channel inhibition using PatchXpress.

PatchXpress, an automated 16-channel parallel patch clamp system, was used to determine inhibition of human ether-a-go-go related gene (hERG) potassium channels by known blockers. A monoclonal cell line stably expressing hERG potassium channels was generated in CHO-K1 cells. Results were compared to conventional patch clamp experiments using similar voltage protocols and solutions. Success rates were evaluated for cell recordings under a variety of conditions, including Accumax versus trypsin treatment to harvest cells, single versus double compound additions, and polystyrene versus glass-coated compound plates. Finally, polystyrene versus glass-coated compound plates were evaluated, and the authors found that for some compounds (but not all), preparation of compound samples in glass-coated plates resulted in inhibition that more closely matched data obtained by conventional experiments.

Systematic approach to interpretation of the lumbar spine MR imaging examination.

The lumbar spine MR imaging study is one of the most frequently ordered MR imaging examinations in the United States. Numerous possible causes exist for low back pain, which contributes to the diagnostic challenge that imagers face in arriving at clinically relevant diagnoses from the anatomic information provided by MR imaging. In this article, the authors suggest a systematic approach to MR imaging interpretation. The evaluation may be altered, based on individual preferences and the specific clinical scenario. The author also highlights commonly encountered disease processes, pertinent MR imaging anatomy, and some common diagnostic pitfalls. Normal and abnormal spine MR images are also shown.

Magnetic resonance imaging of the collateral ligaments and the anatomic quadrants of the knee.

Injuries to the lateral and medial supporting structures of the knee can be significantly disabling and somewhat difficult to detect and evaluate clinically. An accurate imaging evaluation of these structures requires the use of the appropriate MR imaging sequences and the detailed knowledge of the anatomic structures that are present in these locations. Normal function is dependent on the integrity of the complex functional structures and effective clinical treatment, including surgical repair, of these structures is predicated on an optimal diagnostic evaluation. A successful diagnostic evaluation can expedite treatment and provide the best opportunity for a favorable long-term outcome.

Gnathodiaphyseal dysplasia.

Gnathodiaphyseal dysplasia (GDD) is an orthopedic condition involving cemento-osseous lesions of jaw bones, sclerosis, bowing of tubular bones, and overall bone fragility. This is a case report of GDD in which an 18-year-old female presented with a several-year history of bony abnormalities of the jaw and long bones. Radiographs demonstrated endosteal thickening, bowing of long bones, and abnormalities of the skull. These findings, along with the patient's known maxillary region cementoma, led to a diagnosis of GDD.

Infected dermoid sinus tract mimicking recurrent sinusitis in an adolescent male.

Nasal dermoid sinus cysts are the most common congenital midline nasofrontal masses, but they can easily be confused with simple recurrent sinusitis upon clinical presentation. We report the case of a 14-year-old male who was initially diagnosed with frontal sinusitis and treated with antibiotics but, after multiple episodes of recurrence, was later sent for imaging. Computed tomography and magnetic resonance imaging showed an infected dermoid sinus tract. Subsequent surgery removed the mass and pathologic investigation proved the diagnosis.

Bone tumor mimics: avoiding misdiagnosis.

Whether discovered incidentally or as part of a focused diagnostic evaluation, the finding of a benign osseous lesion that has radiologic features resembling a bone tumor is not uncommon. Some of the more common benign and nonneoplastic entities that can sometimes be confused with tumors are the following: cortical desmoid, Brodie abscess, synovial herniation pit, pseudocyst, enostosis, intraosseous ganglion cyst, fibrous dysplasia, stress fracture, avulsion fracture (healing stage), bone infarct, myositis ossificans, brown tumor, and subchondral cyst. Accurate diagnosis and management of these lesions require a basic understanding of their epidemiology, clinical presentations, anatomic distributions, imaging features, differential considerations, and therapeutic options. This in-depth review of 13 potential bone tumor mimics will assist the radiologist in correctly identifying these benign lesions and in avoiding misdiagnosis and related morbidity. This review will also aid the radiologist in making appropriate recommendations to the referring physician for management or further imaging.

Extramedullary intradural spinal tumors: a pictorial review.

Defining the location of tumors and mass lesions of the spine in relation to the spinal cord and the dura is of the utmost importance as certain types of lesions tend to occur in certain locations. The differential diagnostic considerations will vary according to location of the mass lesion as will the treatment and prognosis of these various lesions. The category of extramedullary intradural masses includes a variety of lesions from meningiomas and nerve sheath tumors (neurofibromas, schwannomas) to less common tumors (hemangiopericytoma), metastases, benign tumors (lipoma, dermoid, epidermoid), inflammatory disorders (arachnoid adhesions, sarcoidosis), vascular lesions (spinal-dural arteriovenous fistula), and cystic lesions (perineural or Tarlov cysts). Characteristic magnetic resonance imaging findings are helpful for localization and characterization of these lesions before treatment, as well as for follow-up after treatment. We present a pictorial review of the various extramedullary intradural lesions of the spine, with pathologic correlation. We discuss imaging features that are typical for the various entities and describe various therapeutic options that are important considerations for surgical treatment of these lesions.

Radiology corner. Answer to last month's radiology case and images: Schistosomiasis japonicum involving the liver and colon.

Abdominal radiography showed subtle curvilinear densities overlying the left lower quadrant and central within the pelvis (Fig. 1a). This was confirmed on intravenous contrast-enhanced abdominopelvic CT to be caused by thin mucosal surface calcifications within the descending and sigmoid colons (Fig. 1b, c). Incidental note is made of thin right hepatic lobe capsule calcifications (Fig. 1d). These colonic and hepatic calcifications are characteristic of Schistosomiasis infection of the gastrointestinal tract. Liver biopsy confirmed Schistosomiasis japonicum infection.

Case for diagnosis. Elbow synovial fold syndrome.

The following report reviews presentation, pathophysiology, characteristic imaging findings and current treatment options for elbow synovial fold syndrome. We present the case of a 17 year old female with clinical and MR imaging evidence of this condition and provide a comprehensive review of the limited peer-reviewed literature.

Calcaneal osteochondroma.

Osteochondromas are essentially the most common primary bone tumors. These benign cartilage producing neoplasms are generally asymptomatic and have a relatively small potential for adverse effects. HME, the familial form of this tumor, is associated with a greater incidence of complications, the most significant of which is sarcomatous degeneration to secondary chondrosarcomas. Various imaging techniques can be used to characterize these lesions, but in the absence of symptoms or signs of complications, plain-film radiography is usually sufficient for diagnosis as this tumor has a characteristic radiographic appearance. Once this benign tumor is identified and more serious forms of tumor are ruled out, treatment is generally not needed. If a malignancy is suspected, however, complete surgical excision is the preferred treatment as this usually ensures long term survival.

Disruption of the inferior popliteomeniscal ligamentous fascicle.

The superior popliteomeniscal ligamentous fascicular injury association with lateral meniscal injury has been previously documented in the literature. Both superior and inferior popliteomeniscal ligamentous injuries have demonstrated associations with lateral meniscal motion, and these ligaments can be disrupted in posterolateral corner knee injuries as well. The current case demonstrates an unusual case of isolated inferior popliteomeniscal ligamentous fascicular injury. We examine and discuss its association with the superior popliteomeniscal ligament, posterolateral corner injuries and the fascicular relationship to lateral meniscal tears.

The accuracy of magnetic resonance imaging in the assessment of glenohumeral articular degenerative disease.

Degenerative joint disease is a major source of disability in the world with over 43 million individuals suffering from the affliction in the United States alone. It is the most common cause of activity limitation in individuals over 65 years of age. While much of the focus in recent years has been on osteoarthritis of the hips and knees, shoulder degenerative disease is becoming a more commonly recognized source of morbidity with a wide range of associated lifestyle-limiting disabilities. At the same time therapeutic options for treatment of degenerative joint disease are rapidly increasing, both medically and surgically. This combination of factors makes it necessary to determine a reliable, noninvasive means by which to accurately diagnose the early changes of shoulder degenerative disease. The clinical diagnosis of shoulder osteoarthritis is extremely challenging. There are numerous existing mimickers such as rotator cuff injuries, bursitis, and impingement syndrome. While the conventional radiographic findings are well recognized, they are generally late developments in the course of the disease when therapeutic options are more limited and less effective. Additionally, plain film evaluation has poor sensitivity for the detection of many of the alternative diagnoses that may underlie chronic shoulder pain. Though correlative findings are seen in MR imaging, its role in evaluating glenohumeral degenerative changes has been limited, with much of the focus being on the identification of tendinous and ligamentous disease or osseous tumors. A retrospective analysis is presented which demonstrates the efficacy of MR imaging in assessing GHJ OA, as well as shows that dedicated evaluation for specific degenerative findings results in improved detection rates of GHJ degenerative disease. It is believed that with improved detection and reporting, improved clinical care for this prevalent disorder may be achieved.