Development of a patient specific cartilage graft using magnetic resonance imaging and 3D printing.

OBJECTIVES: The goal of this project was to develop and validate a patient-specific, anatomically correct graft for cartilage restoration using magnetic resonance imaging (MRI) data and 3-dimensional (3D) printing technology. The specific aim was to test the accuracy of a novel method for 3D printing and implanting individualized, anatomically shaped bio-scaffolds to treat cartilage defects in a human cadaveric model. We hypothesized that an individualized, anatomic 3D-printed scaffold designed from MRI data would provide a more optimal fill for a large cartilage defect compared to a generic flat scaffold. METHODS: Four focal cartilage defects (FCDs) were created in paired human cadaver knees, age <40 years, in the weight-bearing surfaces of the medial femoral condyle (MFC), lateral femoral condyle (LFC), patella, and trochlea of each knee. MRIs were obtained, anatomic grafts were designed and 3D printed for the left knee as an experimental group, and generic flat grafts for the right knee as a control group. Grafts were implanted into corresponding defects and fixed using tissue adhesive. Repeat post-implant MRIs were obtained. Graft step-off was measured as the distance in mm between the surface of the graft and the native cartilage surface in a direction perpendicular to the subchondral bone. Graft contour was measured as the gap between the undersurface of the graft and the subchondral bone in a direction perpendicular to the joint surface. RESULTS: Graft step-off was statistically significantly better for the anatomic grafts compared to the generic grafts in the MFC (0.0 â€‹± â€‹0.2 â€‹mm vs. 0.7 â€‹± â€‹0.5 â€‹mm, p â€‹< â€‹0.001), LFC (0.1 â€‹± â€‹0.3 â€‹mm vs. 1.0 â€‹± â€‹0.2 â€‹mm, p â€‹< â€‹0.001), patella (-0.2 â€‹± â€‹0.3 â€‹mm vs. -1.2 â€‹± â€‹0.4 â€‹mm, p â€‹< â€‹0.001), and trochlea (-0.4 â€‹± â€‹0.3 vs. 0.4 â€‹± â€‹0.7, p â€‹= â€‹0.003). Graft contour was statistically significantly better for the anatomic grafts in the LFC (0.0 â€‹± â€‹0.0 â€‹mm vs. 0.2 â€‹± â€‹0.4 â€‹mm, p â€‹= â€‹0.022) and trochlea (0.0 â€‹± â€‹0.0 â€‹mm vs. 1.4 â€‹± â€‹0.7 â€‹mm, p â€‹< â€‹0.001). The anatomic grafts had an observed maximum step-off of -0.9 â€‹mm and a maximum contour mismatch of 0.8 â€‹mm. CONCLUSION: This study validates a process designed to fabricate anatomically accurate cartilage grafts using MRI and 3D printing technology. Anatomic grafts demonstrated superior fit compared to generic flat grafts. LEVEL OF EVIDENCE: Level IV.

Anatomical Differences Between Human and Pig Hearts and Their Relevance for Cardiac Xenotransplantation Surgical Technique.

Cardiac xenotransplantation has been proposed to bridge the gap between supply and demand for patients with end-stage heart failure requiring transplantation. However, differences in pig anatomy compared with human anatomy require modification of the surgical approach. In addition, careful consideration should be given to size matching before transplantation. (Level of Difficulty: Advanced.).

A Novel Approach to Virtual Surgical Planning for Mandibular and Midfacial Reconstruction With a Fibula Free Flap.

BACKGROUND: The fibula free flap is a major workhorse in facial reconstruction. To decrease operative times, virtual surgical planning (VSP) has been implemented. Traditional VSP is costly and may delay operative planning. in this study, the authors present a novel algorithm using readily accessible software packages to perform VSP in a manner that is quick and cost-effective. METHODS: A 6-part survey was administered to physicians with prior training in facial reconstruction. Fourteen physicians participated regarding outcomes on 10 patients who underwent mandibular or midfacial fibula free flap reconstruction. Participants were asked to match the true postoperative and VSP models and rate the similarity of the models using the Likert scale (0-10). The goal was to determine whether the VSP models accurately depicted the actual reconstruction, and whether they would use VSP in future clinical practice. RESULTS: The physicians surveyed were able to match the models correctly 93.6% of the time. The mean score for the similarity between virtual and actual models ranged between 7.60 and 8.80. Most participants (90.9%) who answered stated they would use our VSP algorithm if they were trained in the protocol. CONCLUSIONS AND RELEVANCE: Virtual surgical planning models were created utilizing our novel algorithm. Participants matched the preoperative VSP plan with the postoperative model most of the time and rated the similarity well. Participants in our study are interested in learning more about physician performed VSP. The authors believe this model may be financially and clinically relevant and serve as an excellent educational tool.

A Mass Spectrometric Assay of METTL3/METTL14 Methyltransferase Activity.

A variety of covalent modifications of RNA have been identified and demonstrated to affect RNA processing, stability, and translation. Methylation of adenosine at the N6 position (m6A) in messenger RNA (mRNA) is currently the most well-studied RNA modification and is catalyzed by the RNA methyltransferase complex METTL3/METTL14. Once generated, m6A can modulate mRNA splicing, export, localization, degradation, and translation. Although potent and selective inhibitors exist for several members of the Type I S-adenosylmethionine (SAM)-dependent methyltransferase family, no inhibitors have been reported for METTL3/METTL14 to date. To facilitate drug discovery efforts, a sensitive and robust mass spectrometry-based assay for METTL3/METTL14 using self-assembled monolayer desorption/ionization (SAMDI) technology has been developed. The assay uses an 11-nucleotide single-stranded RNA compared to a previously reported 27-nucleotide substrate. IC50 values of mechanism-based inhibitors S-adenosylhomocysteine (SAH) and sinefungin (SFG) are comparable between the SAMDI and radiometric assays that use the same substrate. This work demonstrates that SAMDI technology is amenable to RNA substrates and can be used for high-throughput screening and compound characterization for RNA-modifying enzymes.

Deciphering the Allosteric Binding Mechanism of the Human Tropomyosin Receptor Kinase A ( hTrkA) Inhibitors.

Access to cryptic binding pockets or allosteric sites on a kinase that present themselves when the enzyme is in a specific conformational state offers a paradigm shift in designing the next generation small molecule kinase inhibitors. The current work showcases an extensive and exhaustive array of in vitro biochemical and biophysical tools and techniques deployed along with structural biology efforts of inhibitor-bound kinase complexes to characterize and confirm the cryptic allosteric binding pocket and docking mode of the small molecule actives identified for hTrkA. Specifically, assays were designed and implemented to lock the kinase in a predominantly active or inactive conformation and the effect of the kinase inhibitor probed to understand the hTrkA binding and hTrkB selectivity. The current outcome suggests that inhibitors with a fast association rate take advantage of the inactive protein conformation and lock the kinase state by also exhibiting a slow off-rate. This in turn shifts the inactive/active state protein conformational equilibrium cycle, affecting the subsequent downstream signaling.

New and emerging patient-centered CT imaging and image-guided treatment paradigms for maxillofacial trauma.

This article reviews the conceptual framework, available evidence, and practical considerations pertaining to nascent and emerging advances in patient-centered CT-imaging and CT-guided surgery for maxillofacial trauma. These include cinematic rendering-a novel method for advanced 3D visualization, incorporation of quantitative CT imaging into the assessment of orbital fractures, low-dose CT imaging protocols made possible with contemporary scanners and reconstruction techniques, the rapidly growing use of cone-beam CT, virtual fracture reduction with design software for surgical pre-planning, the use of 3D printing for fabricating models and implants, and new avenues in CT-guided computer-aided surgery.

Selective inhibition of the p38α MAPK-MK2 axis inhibits inflammatory cues including inflammasome priming signals.

p38α activation of multiple effectors may underlie the failure of global p38α inhibitors in clinical trials. A unique inhibitor (CDD-450) was developed that selectively blocked p38α activation of the proinflammatory kinase MK2 while sparing p38α activation of PRAK and ATF2. Next, the hypothesis that the p38α-MK2 complex mediates inflammasome priming cues was tested. CDD-450 had no effect on NLRP3 expression, but it decreased IL-1ß expression by promoting IL-1ß mRNA degradation. Thus, IL-1ß is regulated not only transcriptionally by NF-κB and posttranslationally by the inflammasomes but also posttranscriptionally by p38α-MK2. CDD-450 also accelerated TNF-α and IL-6 mRNA decay, inhibited inflammation in mice with cryopyrinopathy, and was as efficacious as global p38α inhibitors in attenuating arthritis in rats and cytokine expression by cells from patients with cryopyrinopathy and rheumatoid arthritis. These findings have clinical translation implications as CDD-450 offers the potential to avoid tachyphylaxis associated with global p38α inhibitors that may result from their inhibition of non-MK2 substrates involved in antiinflammatory and housekeeping responses.

3D Printing Prototypes for Healthcare Professionals: Creating a Reciprocating Syringe.

3D printing (additive manufacturing) has been around since 1984, but interest in the technology has increased exponentially as it has become both accessible and inexpensive. The applications of the technology in healthcare are still being explored; however, initial forays have been encouraging. It has the potential to revolutionize the process of prototyping for healthcare professionals by democratizing the process and enhancing collaboration, making it cheaper to do iterative prototyping with little or no engineering experience. This case report details the creation of a multi-lumen reciprocating syringe with 3D printing. The product has been created and tested using a variety of publicly available resources. It provides a detailed overview of the approach and the framework required to create such a medical device. However, the implications of this report are much larger than this one product, and the fundamental ideas discussed here could be used for creating customized solutions for many healthcare problems.

Surgical reconstruction of the ossicular chain with custom 3D printed ossicular prosthesis.

BACKGROUND: Conductive hearing loss due to ossicular abnormalities occurs from many causes, including trauma, infection, cholesteatoma, surgery and congenital anomalies. Surgical reconstruction of the ossicular chain is a well-established procedure for repair of ossicular defects, but is still plagued by high failure rates. Underlying disease and proper sizing of prostheses are two challenges that lead to component failure. Three-dimensional (3D) printing has been used successfully to solve a number of medical prosthesis problems. Custom 3D printing an individualized ossicular prosthesis would be a potential solution for the wide range of anatomic variation encountered in the pathological middle ear, and could decrease the rate of post-operative prosthesis displacement by increasing the likelihood of a proper fit, in addition to decreasing surgical time.In this study, the incus was removed from three formalin-fixed cadaveric human temporal bones with no macro- or microscopic evidence of pathology. Imaging of the cadaveric bone was obtained using a standard temporal bone CT protocol. A custom prosthesis for each cadaveric human temporal bone was designed using the Mimics Innovation Suite software (Materialise, Belgium) and fabricated on a Form2 3D printer (FormLabs, Somerville, Massachusetts). Four surgeons then performed insertion of each prosthesis into each middle ear, blinded to the bone from and for which each was designed. The surgeons were asked to match each prosthesis to its correct parent bone. RESULTS: Each prosthesis had unique measurements. Each of the four surgeons was able to correctly match the prosthesis model to its intended temporal bone. The chances of this occurring randomly are 1:1296. CONCLUSIONS: A custom 3D printed ossicular prosthesis is a viable solution for conductive hearing loss due to ossicular chain defects. Commercially available CT scanners can detect significant anatomic differences in normal human middle ear ossicles. These differences can be accurately represented with current 3D printing technology and, more significantly, surgeons can detect these differences.

We Built This House; It's Time to Move in: Leveraging Existing DICOM Structure to More Completely Utilize Readily Available Detailed Contrast Administration Information.

The Digital Imaging and Communications in Medicine (DICOM) standard is the universal format for interoperability in medical imaging. In addition to imaging data, DICOM has evolved to support a wide range of imaging metadata including contrast administration data that is readily available from many modern contrast injectors. Contrast agent, route of administration, start and stop time, volume, flow rate, and duration can be recorded using DICOM attributes [1]. While this information is sparsely and inconsistently recorded in routine clinical practice, it could potentially be of significant diagnostic value. This work will describe parameters recorded by automatic contrast injectors, summarize the DICOM mechanisms available for tracking contrast injection data, and discuss the role of such data in clinical radiology.

Differential kinetics and inhibition of purified recombinant tyrosine kinase 2 (TYK-2) and its catalytic domain JH-1.

The Janus kinase (JAK) family consists of four members: JAK-1, -2, -3 and tyrosine kinase 2 (TYK-2). Recent work suggests that cytokine signaling through TYK-2 may play a critical role in a number of inflammatory processes. We recently described the purification and characterization of phosphorylated isoforms of the TYK-2 kinase domain (TYK-2 KD) and its high resolution 3D structure in the presence of inhibitors. We now report the expression and a two-step purification procedure for the doubly tagged full-length construct, H6-FL-TYK-2-FLAG, and examine its properties compared to those of TYK-2 KD. In the presence of ATP and a peptide substrate, H6-FL-TYK-2-FLAG showed a marked lag in phosphopeptide product formation, while TYK-2 KD showed no such lag. This lag could be eliminated by ATP pretreatment, suggesting that the H6-FL-TYK-2-FLAG enzyme was activated by phosphorylation. The potencies of several nanomolar inhibitors were similar for TYK-2 KD and H6-FL-TYK-2-FLAG. However, these same inhibitors were about 1000 times less potent inhibiting the autophosphorylation of H6-FL-TYK-2-FLAG than they were inhibiting the phosphorylation of a peptide substrate modeled after the activation loop sequence of TYK-2. This intriguing result suggests that autophosphorylation and, thus, activation of H6-FL-TYK-2-FLAG is relatively insensitive to inhibition and that present inhibitors act to inhibit TYK-2 subsequent to activation. Inhibition of TYK-2 autophosphorylation may represent a new area of investigation for the JAK family.

Design, synthesis and activity of a potent, selective series of N-aryl pyridinone inhibitors of p38 kinase.

A series of N-aryl pyridinone inhibitors of p38 mitogen activated protein (MAP) kinase were designed and prepared based on the screening hit SC-25028 (1) and structural comparisons to VX-745 (5). The focus of the investigation targeted the dependence of potency and metabolic stability on the benzyloxy connectivity, the role of the C-6 position and the substitution pattern on the N-phenyl ring. Further optimization produced the highly selective and potent pyridinones 2 and 3. These inhibitors exhibited activity in both acute and chronic models of inflammation.

Discovery of PH-797804, a highly selective and potent inhibitor of p38 MAP kinase.

The synthesis and SAR studies of a novel N-aryl pyridinone class of p38 kinase inhibitors are described. Systematic structural modifications to the HTS lead, 5, led to the identification of (-)-4a as a clinical candidate for the treatment of inflammatory diseases. Additionally, the chiral synthesis and properties of (-)-4a are described.

Anti-inflammatory activity and neutrophil reductions mediated by the JAK1/JAK3 inhibitor, CP-690,550, in rat adjuvant-induced arthritis.

BACKGROUND: The Janus kinase (JAK) family of tyrosine kinases includes JAK1, JAK2, JAK3 and TYK2, and is required for signaling through Type I and Type II cytokine receptors. CP-690,550 is a potent and selective JAK inhibitor currently in clinical trials for rheumatoid arthritis (RA) and other autoimmune disease indications. In RA trials, dose-dependent decreases in neutrophil counts (PBNC) were observed with CP-690,550 treatment. These studies were undertaken to better understand the relationship between JAK selectivity and PBNC decreases observed with CP-690,550 treatment. METHODS: Potency and selectivity of CP-690,550 for mouse, rat and human JAKs was evaluated in a panel of in vitro assays. The effect of CP-690,550 on granulopoiesis from progenitor cells was also assessed in vitro using colony forming assays. In vivo the potency of orally administered CP-690,550 on arthritis (paw edema), plasma cytokines, PBNC and bone marrow differentials were evaluated in the rat adjuvant-induced arthritis (AIA) model. RESULTS: CP-690,550 potently inhibited signaling through JAK1 and JAK3 with 5-100 fold selectivity over JAK2 in cellular assays, despite inhibiting all four JAK isoforms with nM potency in in vitro enzyme assays. Dose-dependent inhibition of paw edema was observed in vivo with CP-690,550 treatment. Plasma cytokines (IL-6 and IL-17), PBNC, and bone marrow myeloid progenitor cells were elevated in the context of AIA disease. At efficacious exposures, CP-690,550 returned all of these parameters to pre-disease levels. The plasma concentration of CP-690,550 at efficacious doses was above the in vitro whole blood IC50 of JAK1 and JAK3 inhibition, but not that of JAK2. CONCLUSION: Results from this investigation suggest that CP-690,550 is a potent inhibitor of JAK1 and JAK3 with potentially reduced cellular potency for JAK2. In rat AIA, as in the case of human RA, PBNC were decreased at efficacious exposures of CP-690,550. Inflammatory end points were similarly reduced, as judged by attenuation of paw edema and cytokines IL-6 and IL-17. Plasma concentration at these exposures was consistent with inhibition of JAK1 and JAK3 but not JAK2. Decreases in PBNC following CP-690,550 treatment may thus be related to attenuation of inflammation and are likely not due to suppression of granulopoiesis through JAK2 inhibition.

Structural and thermodynamic characterization of the TYK2 and JAK3 kinase domains in complex with CP-690550 and CMP-6.

Janus kinases (JAKs) are critical regulators of cytokine pathways and attractive targets of therapeutic value in both inflammatory and myeloproliferative diseases. Although the crystal structures of active JAK1 and JAK2 kinase domains have been reported recently with the clinical compound CP-690550, the structures of both TYK2 and JAK3 with CP-690550 have remained outstanding. Here, we report the crystal structures of TYK2, a first in class structure, and JAK3 in complex with PAN-JAK inhibitors CP-690550 ((3R,4R)-3-[4-methyl-3-[N-methyl-N-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]piperidin-1-yl]-3-oxopropionitrile) and CMP-6 (tetracyclic pyridone 2-t-butyl-9-fluoro-3,6-dihydro-7H-benz[h]-imidaz[4,5-f]isoquinoline-7-one), both of which bind in the ATP-binding cavities of both JAK isozymes in orientations similar to that observed in crystal structures of JAK1 and JAK2. Additionally, a complete thermodynamic characterization of JAK/CP-690550 complex formation was completed by isothermal titration calorimetry, indicating the critical role of the nitrile group from the CP-690550 compound. Finally, computational analysis using WaterMap further highlights the critical positioning of the CP-690550 nitrile group in the displacement of an unfavorable water molecule beneath the glycine-rich loop. Taken together, the data emphasize the outstanding properties of the kinome-selective JAK inhibitor CP-690550, as well as the challenges in obtaining JAK isozyme-selective inhibitors due to the overall structural and sequence similarities between the TYK2, JAK1, JAK2 and JAK3 isozymes. Nevertheless, subtle amino acid variations of residues lining the ligand-binding cavity of the JAK enzymes, as well as the global positioning of the glycine-rich loop, might provide the initial clues to obtaining JAK-isozyme selective inhibitors.

Expression, purification, and characterization of TYK-2 kinase domain, a member of the Janus kinase family.

The Janus kinase family consists of four members: JAK-1, -2, -3 and TYK-2. While JAK-2 and JAK-3 have been well characterized biochemically, there is little data on TYK-2. Recent work suggests that TYK-2 may play a critical role in the development of a number of inflammatory processes. We have carried out a series of biochemical studies to better understand TYK-2 enzymology and its inhibition profile, in particular how the TYK-2 phosphorylated forms differ from each other and from the other JAK family members. We have expressed and purified milligram quantities of the TYK-2 kinase domain (KD) to high purity and developed a method to separate the non-, mono- (pY(1054)) and di-phosphorylated forms of the enzyme. Kinetic studies (k(cat(app))/K(m(app))) indicated that phosphorylation of the TYK-2-KD (pY(1054)) increased the catalytic efficiency 4.4-fold compared to its non-phosphorylated form, while further phosphorylation to generate the di-phosphorylated enzyme imparted no further increase in activity. These results are in contrast to those obtained with the JAK-2-KD and JAK-3-KD, where little or no increase in activity occurred upon mono-phosphorylation, while di-phosphorylation resulted in a 5.1-fold increase in activity for the JAK-2-KD. Moreover, ATP-competitive inhibitors demonstrated 10-30-fold shifts in potency (K(i(app))) as a result of the TYK-2-KD phosphorylation state, while the shifts for JAK-3-KD were only 2-3-fold and showed little or no change for JAK-2-KD. Thus, the phosphorlyation state imparted differential effects on both activity and inhibition within the JAK family of kinases.

A benzothiophene inhibitor of mitogen-activated protein kinase-activated protein kinase 2 inhibits tumor necrosis factor alpha production and has oral anti-inflammatory efficacy in acute and chronic models of inflammation.

Activation of the p38 kinase pathway in immune cells leads to the transcriptional and translational regulation of proinflammatory cytokines. Mitogen-activated protein kinase-activated protein kinase 2 (MK2), a direct downstream substrate of p38 kinase, regulates lipopolysaccharide (LPS)-stimulated tumor necrosis factor alpha (TNFalpha) and interleukin-6 (IL-6) production through modulating the stability and translation of these mRNAs. Developing small-molecule inhibitors of MK2 may yield anti-inflammatory efficacy with a different safety profile relative to p38 kinase inhibitors. This article describes the pharmacologic properties of a benzothiophene MK2 inhibitor, PF-3644022 [(10R)-10-methyl-3-(6-methylpyridin-3-yl)-9,10,11,12-tetrahydro-8H-[1,4]diazepino[5',6':4,5]thieno[3,2-f]quinolin-8-one]. PF-3644022 is a potent freely reversible ATP-competitive compound that inhibits MK2 activity (K(i) = 3 nM) with good selectivity when profiled against 200 human kinases. In the human U937 monocytic cell line or peripheral blood mononuclear cells, PF-3644022 potently inhibits TNFalpha production with similar activity (IC(50) = 160 nM). PF-3644022 blocks TNFalpha and IL-6 production in LPS-stimulated human whole blood with IC(50) values of 1.6 and 10.3 microM, respectively. Inhibition of TNFalpha in U937 cells and blood correlates closely with inhibition of phospho-heat shock protein 27, a target biomarker of MK2 activity. PF-3644022 displays good pharmacokinetic parameters in rats and is orally efficacious in both the rat acute LPS-induced TNFalpha model and the chronic streptococcal cell wall-induced arthritis model. Dose-dependent inhibition of TNFalpha production in the acute model and inhibition of paw swelling in the chronic model is observed with ED(50) values of 6.9 and 20 mg/kg, respectively. PF-3644022 efficacy in the chronic inflammation model is strongly correlated with maintaining a C(min) higher than the EC(50) measured in the rat LPS-induced TNFalpha model.

Expression, purification, characterization and crystallization of non- and phosphorylated states of JAK2 and JAK3 kinase domain.

Janus-associated kinases (JAKs) play critical roles in cytokine signaling, and have emerged as viable therapeutic targets in inflammation and oncology related diseases. To date, targeting JAK proteins with highly selective inhibitor compounds have remained elusive. We have expressed the active kinase domains for both JAK2 and JAK3 and devised purification protocols to resolve the non-, mono- (Y1007) and diphosphorylated (Y1007 and Y1008) states of JAK2 and non- and monophosphorylated states of JAK3 (Y980). An optimal purified protein yield of 20, 29 and 69mg per 20L cell culture was obtained for the three JAK2 forms, respectively, and 12.2 and 2.3mg per 10L fermentation for the two JAK3 forms allowing detailed biochemical and biophysical studies. To monitor the purification process we developed a novel HPLC activity assay where a sequential order of phosphorylation was observed whereby the first tyrosine residue was completely phosphorylated prior to phosphorylation of the tandem tyrosine residue. A Caliper-based microfluidics assay was used to determine the kinetic parameters (K(m) and k(cat)) for each phosphorylated state, showing that monophosphorylated (Y1007) JAK2 enzyme activity increased 9-fold over that of the nonphosphorylated species, and increased an additional 6-fold for the diphosphorylated (Y1007/Y1008) species, while phosphorylation of JAK3 resulted in a negligible increase in activity. Moreover, crystal structures have been generated for each isolated state of JAK2 and JAK3 with resolutions better than 2.4A. The generation of these reagents has enabled kinetic and structural characterization to inform the design of potent and selective inhibitors of the JAK family.

Anti-inflammatory properties of a novel N-phenyl pyridinone inhibitor of p38 mitogen-activated protein kinase: preclinical-to-clinical translation.

Signal transduction through the p38 mitogen-activated protein (MAP) kinase pathway is central to the transcriptional and translational control of cytokine and inflammatory mediator production. p38 MAP kinase inhibition hence constitutes a promising therapeutic strategy for treatment of chronic inflammatory diseases, based upon its potential to inhibit key pathways driving the inflammatory and destructive processes in these debilitating diseases. The present study describes the pharmacological properties of the N-phenyl pyridinone p38 MAP kinase inhibitor benzamide [3- [3-bromo-4-[(2,4-difluorophenyl)methoxy]-6-methyl-2- oxo-1(2H)-pyridinyl]-N,4-dimethyl-, (-)-(9CI); PH-797804]. PH-797804 is an ATP-competitive, readily reversible inhibitor of the alpha isoform of human p38 MAP kinase, exhibiting a K(i) = 5.8 nM. In human monocyte and synovial fibroblast cell systems, PH-797804 blocks inflammation-induced production of cytokines and proinflammatory mediators, such as prostaglandin E(2), at concentrations that parallel inhibition of cell-associated p38 MAP kinase. After oral dosing, PH-797804 effectively inhibits acute inflammatory responses induced by systemically administered endotoxin in both rat and cynomolgus monkeys. Furthermore, PH-797804 demonstrates robust anti-inflammatory activity in chronic disease models, significantly reducing both joint inflammation and associated bone loss in streptococcal cell wall-induced arthritis in rats and mouse collagen-induced arthritis. Finally, PH-797804 reduced tumor necrosis factor-alpha and interleukin-6 production in clinical studies after endotoxin administration in a dose-dependent manner, paralleling inhibition of the target enzyme. Low-nanomolar biochemical enzyme inhibition potency correlated with p38 MAP kinase inhibition in human cells and in vivo studies. In addition, a direct correspondence between p38 MAP kinase inhibition and anti-inflammatory activity was observed with PH-797804, thus providing confidence in dose projections for further human studies in chronic inflammatory disease.