The novel kinase inhibitor PRT062070 (Cerdulatinib) demonstrates efficacy in models of autoimmunity and B-cell cancer.

The heterogeneity and severity of certain autoimmune diseases and B-cell malignancies warrant simultaneous targeting of multiple disease-relevant signaling pathways. Dual inhibition of spleen tyrosine kinase (SYK) and Janus kinase (JAK) represents such a strategy and may elicit several benefits relative to selective kinase inhibition, such as gaining control over a broader array of disease etiologies, reducing probability of selection for bypass disease mechanisms, and the potential that an overall lower level suppression of individual targets may be sufficient to modulate disease activity. To this end, we provide data on the discovery and preclinical development of PRT062070 [4-(cyclopropylamino)-2-({4-[4-(ethylsulfonyl)piperazin-1-yl]phenyl}amino)pyrimidine-5-carboxamide hydrochloride], an orally active kinase inhibitor that demonstrates activity against SYK and JAK. Cellular assays demonstrated specific inhibitory activity against signaling pathways that use SYK and JAK1/3. Limited inhibition of JAK2 was observed, and PRT062070 did not inhibit phorbol 12-myristate 13-acetate-mediated signaling or activation in B and T cells nor T-cell antigen receptor-mediated signaling in T cells, providing evidence for selectivity of action. Potent antitumor activity was observed in a subset of B-cell lymphoma cell lines. After oral dosing, PRT062070 suppressed inflammation and autoantibody generation in a rat collagen-induced arthritis model and blocked B-cell activation and splenomegaly in a mouse model of chronic B-cell antigen receptor stimulation. PRT062070 is currently under evaluation in a phase I dose escalation study in patients with B-cell leukemia and lymphoma (NCT01994382), with proof-of-concept studies in humans planned to assess therapeutic potential in autoimmune and malignant diseases.

The selective SYK inhibitor P505-15 (PRT062607) inhibits B cell signaling and function in vitro and in vivo and augments the activity of fludarabine in chronic lymphocytic leukemia.

B-cell receptor (BCR) associated kinases including spleen tyrosine kinase (SYK) contribute to the pathogenesis of B-cell malignancies. SYK is persistently phosphorylated in a subset of non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL), and SYK inhibition results in abrogation of downstream kinase activity and apoptosis. P505-15 (also known as PRT062607) is a novel, highly selective, and orally bioavailable small molecule SYK inhibitor (SYK IC(50) = 1 nM) with anti-SYK activity that is at least 80-fold greater than its affinity for other kinases. We evaluated the preclinical characteristics of P505-15 in models of NHL and CLL. P505-15 successfully inhibited SYK-mediated B-cell receptor signaling and decreased cell viability in NHL and CLL. Oral dosing in mice prevented BCR-mediated splenomegaly and significantly inhibited NHL tumor growth in a xenograft model. In addition, combination treatment of primary CLL cells with P505-15 plus fludarabine produced synergistic enhancement of activity at nanomolar concentrations. Our findings support the ongoing development of P505-15 as a therapeutic agent for B-cell malignancies. A dose finding study in healthy volunteers has been completed.

Specific inhibition of spleen tyrosine kinase suppresses leukocyte immune function and inflammation in animal models of rheumatoid arthritis.

Based on genetic studies that establish the role of spleen tyrosine kinase (Syk) in immune function, inhibitors of this kinase are being investigated as therapeutic agents for inflammatory diseases. Because genetic studies eliminate both adapter functions and kinase activity of Syk, it is difficult to delineate the effect of kinase inhibition alone as would be the goal with small-molecule kinase inhibitors. We tested the hypothesis that specific pharmacological inhibition of Syk activity retains the immunomodulatory potential of Syk genetic deficiency. We report here on the discovery of (4-(3-(2H-1,2,3-triazol-2-yl)phenylamino)-2-((1R,2S)-2-aminocyclohexylamino) pyrimidine-5-carboxamide acetate (P505-15), a highly specific and potent inhibitor of purified Syk (IC50 1-2 nM). In human whole blood, P505-15 potently inhibited B cell antigen receptor-mediated B cell signaling and activation (IC50 0.27 and 0.28 µM, respectively) and Fcε receptor 1-mediated basophil degranulation (IC50 0.15 µM). Similar levels of ex vivo inhibition were measured after dosing in mice (Syk signaling IC50 0.32 µM). Syk-independent signaling and activation were unaffected at much higher concentrations, demonstrating the specificity of kinase inhibition in cellular systems. Oral administration of P505-15 produced dose-dependent anti-inflammatory activity in two rodent models of rheumatoid arthritis. Statistically significant efficacy was observed at concentrations that specifically suppressed Syk activity by ∼67%. Thus specific Syk inhibition can mimic Syk genetic deficiency to modulate immune function, providing a therapeutic strategy in P505-15 for the treatment of human diseases.

Review of continuing education course on hemostasis.

The continuing education course "Hemostasis" provided a comprehensive review of hemostasis and selected perturbations of the underlying processes as well as an assessment of hemostasis in animal models and preclinical testing environments. The session began with a review of the current state of understanding of hemostasis and how the waterfall or cascade of activation has transformed to the current cell-based, membrane-associated sequence of highly regulated events. The specific mechanisms of drug-induced thrombocytopenia were then presented, followed by a discussion of the relationships of coagulation and platelets in inflammation and cancer metastasis and platelet activity. Evaluation of hemostasis and platelet function in animals and especially in the environment of the contract research facility concluded the session.

Ceramic-on-ceramic total hip arthroplasty.

Ceramic bearing surfaces have been introduced to prevent bone loss after osteolysis seen with conventional polyethylene bearing surfaces. One hundred three ceramic-on-ceramic total hip arthroplasties in 97 patients were retrospectively reviewed. Average follow-up was 50.4 months. Preoperative Harris Hip Score was 49.5 points, which improved to 87.2 postoperatively (P < .05). Pain score improved from 13.7 points preoperatively to 40.6 points postoperatively (P < .05). Functional score improved from 30 points preoperatively to 41 points postoperatively (P < .05). No fractures, dislocations, infections, or osteolysis was observed on radiographs. Five patients (4.9%), at 11, 16, 30, 38, and 60 months postoperatively, presented with "squeaky" hips that continue to perform well. Long-term studies will be required to determine the true efficacy of these hard bearing surfaces.

Decreased dislocation after revision total hip arthroplasty using larger femoral head size and posterior capsular repair.

The purpose of this study was to determine if the use of both a larger femoral head size and a posterior capsular repair would lead to a decreased incidence of dislocation following revision total hip arthroplasty (THA). Two hundred forty-two consecutive revision THAs with posterolateral approach were performed between 2000 and 2005. Group 1 had 132 revision THAs with posterolateral approach and 28-mm head size without posterior capsule repair. Group 2 had 100 revision THAs with a 32-mm head size and repair of the remaining hip capsule. There were no statistically significant differences in the two groups. Group 1 had 14 dislocations (10.6%). Group 2 had 3 dislocations (2.7%) (P < .05). Based on the results of this retrospective review, the authors recommend the use of both larger femoral head sizes and repair of any posterior capsular tissue available in patients undergoing revision hip arthroplasty.

Acetabular component revision using a porous tantalum biomaterial: a case series.

Biologic ingrowth can be difficult to achieve in acetabular component revision, especially in cases with significant bone loss. The purpose of this study was to review our clinical results of acetabular component revisions in patients with significant bone loss using a porous tantalum biomaterial. This is a retrospective review of 25 patients. There were 16 females and 9 males with a mean age of 71.7 +/- 10.54 years. The mean follow up was 39 +/- 11.09 months (range, 28-55 months). All patients in this series had combined segmental and cavitary bone loss, Paprosky type 2 or type 3. Of 22 patients in this series, 21 had a well-fixed and functioning implant at latest follow up. All 21 patients developed ingrowth along the tantalum surface despite compromised host bone. There were no cases of dislocation or aseptic loosening. Porous tantalum appears to be a promising material for use in revision hip arthroplasty to facilitate biologic ingrowth in patients with acetabular bone loss.

Fulkerson procedure for chronic patella component dislocation after total knee arthroplasty.

Chronic patella component dislocation after primary knee arthroplasty can be a challenging problem. The purpose of this case series was to review our results of a chronic patella dislocation after total knee arthroplasty treated with a Fulkerson procedure (anteromedialization of the tibial tubercle). Five patients with an average age of 68.8 years (range, 60-76 years) underwent a Fulkerson procedure to restore extensor mechanism alignment. The tibial tubercle osteotomy was secured with lag screws with an average medialization of approximately 1.5 cm. The average preoperative Knee Society score was 70.5 points that improved to 85 points postoperatively. The average knee flexion was 93 degrees preoperatively that improved to 101 degrees postoperatively. Adequate patellofemoral alignment was achieved in all patients along with union of the tibial tubercle osteotomy site. The Fulkerson procedure appears to be an excellent treatment option in patients with a chronically dislocated patella component after total knee arthroplasty.

Sites of modification of hemospan, a poly(ethylene glycol)-modified human hemoglobin for use as an oxygen therapeutic.

Hemospan is an acellular hemoglobin-based oxygen therapeutic in clinical trials in Europe and the United States. The product is prepared by site-specific conjugation of maleimide-activated poly(ethylene) glycol (PEG, MW approximately 5500) to human oxyhemoglobin through maleimidation reactions either (1) directly to reactive Cys thiols or (2) at surface Lys groups following thiolation using 2-iminothiolane. The thiolation/maleimidation reactions lead to the addition of approximately 8 PEGs per hemoglobin tetramer. Identification of PEG modified globins by SDS-PAGE and MALDI-TOF reveals a small percentage of protein migrating at the position for unmodified globin chains and the remaining as separate bands representing globin chains conjugated with 1 to 4 PEGs per chain. Identification of PEG modification sites on individual alpha and beta globins was made using reverse-phase HPLC, showing a series of alpha globins conjugated with 0 to 3 PEGs and a series of beta globins conjugated with 0 to 4 PEGs per globin. Mass analysis of tryptic peptides from hemoglobin thiolated and maleimidated with N-ethyl maleimide showed the same potential sites of modification regardless of thiolation reaction ratio, with seven sites identified on beta globins at beta8, beta17, beta59, beta66, beta93, beta95, and beta132 and three sites identified on alpha globins at alpha7, alpha16, and alpha40.

Solution structure of poly(ethylene) glycol-conjugated hemoglobin revealed by small-angle X-ray scattering: implications for a new oxygen therapeutic.

Developing protein therapeutics has posed challenges due to short circulating times and toxicities. Recent advances using poly(ethylene) glycol (PEG) conjugation have improved their performance. A PEG-conjugated hemoglobin (Hb), Hemospan, is in clinical trials as an oxygen therapeutic. Solutions of PEG-hemoglobin with two (P5K2) or six to seven strands of 5-kD PEG (P5K6) were studied by small-angle x-ray scattering. PEGylation elongates the dimensions (Hb < P5K2 < P5K6) and leaves the tertiary hemoglobin structure unchanged but compacts its quaternary structure. The major part of the PEG chains visualized by ab initio reconstruction protrudes away from hemoglobin, whereas the rest interacts with the protein. PEGylation introduces intermolecular repulsion, increasing with conjugated PEG amount. These results demonstrate how PEG surface shielding and intermolecular repulsion may prolong intravascular retention and lack of reactivity of PEG-Hb, possibly by inhibiting binding to the macrophage CD163 hemoglobin-scavenger receptor. The proposed methodology for assessment of low-resolution structures and interactions is a powerful means for rational design of PEGylated therapeutic agents.

A quantitative framework for the design of acellular hemoglobins as blood substitutes: implications of dynamic flow conditions.

The delivery of oxygen to tissue by cell-free carriers eliminates intraluminal barriers associated with red blood cells. This is important in arterioles, since arteriolar tone controls capillary perfusion. We describe a mathematical model for O(2) transport by hemoglobin solutions and red blood cells flowing through arteriolar-sized tubes to optimize values of p50, Hill number, hemoglobin molecular diffusivity and concentration. Oxygen release is evaluated by including an extra-luminal resistance term to reflect tissue oxygen consumption. For low consumption (i.e., high resistance to O(2) release) a hemoglobin solution with p50=15 mmHg, n=1, D(HBO2)=3 x 10(-7) cm(2)/s delivers O(2) at a rate similar to that of red blood cells. For high consumption, the p50 must be decreased to 5 mmHg. The model predicts that regardless of size, hemoglobin solutions with higher p50 will present excess O(2) to arteriolar walls. Oversupply of O(2) to arteriolar walls may cause constriction and paradoxically reduced capillary perfusion.

Venezuelan equine encephalitis virus vaccine candidate (V3526) safety, immunogenicity and efficacy in horses.

A new vaccine, V3526, is a live-attenuated virus derived by site-directed mutagenesis from a virulent clone of the Venezuelan equine encephalitis virus (VEEV) IA/B Trinidad donkey (TrD) strain, intended for human use in protection against Venezuelan equine encephalitis (VEE). Two studies were conducted in horses to evaluate the safety, immunogenicity, ability to boost and protective efficacy of V3526 against challenges of TrD and VEEV IE 64A99. Horses were vaccinated subcutaneously (SC) with 10(7), 10(5), 10(3) or 10(2) plaque-forming units (pfu) of V3526. Control horses were sham immunized. In the first study, challenge viruses (TrD or 64A99) were administered SC 28 days post-vaccination (PV). No viremia and only mild fluctuation in white blood cell counts were observed PV. None of the V3526 vaccinated horses showed clinical signs of disease or pathology of VEE post-challenge (PC). In contrast, control horses challenged SC with 10(4)pfu TrD became viremic and showed classical signs of VEE beginning on Day 3 PC, including elevated body temperature, anorexia, leukopenia and malaise. Moderate to severe encephalitis was found in three of five control horses challenged with TrD. Control horses challenged with 64A99 failed to develop detectable viremia, but did exhibit a brief febrile episode at 1-3 days PC. None of the 10 immunized horses challenged with 64A99 became pyrexic. Twenty four of 25 horses immunized with V3526 in the first study developed serum neutralizing antibody to TrD and 64A99 within 14 days PV. Vaccinations with V3526, at doses as low as 10(2)pfu, were safe and efficacious in protecting horses against a virulent TrD virus challenge. The second study supported that repeat dosing resulted in an increase in serum neutralizing antibody to TrD.

Impaction grafting for femoral component revision using a non-polished bead-blasted chrome cobalt stem-average 8 1/2-year follow-up.

Impaction grafting for femoral component revision in patients with significant bone loss has been reported using a tapered polished femoral component that is meant to subside. This study reports our results of femoral component revision using impaction grafting with a bead-blasted chrome cobalt stem designed not to subside. Forty-eight femoral component revisions using impaction grafting were retrospectively reviewed with a minimum 6 1/2-year follow-up. There were 2 failures due to aseptic loosening of the femoral component (4%). There were 22 total complications, and the overall failure rate was 21%. Impaction grafting for femoral component revision using a bead-blasted chrome cobalt stem in patients with a large femoral canal diameter has shown good results with respect to aseptic loosening with minimal subsidence.

Transfer of the long head of the triceps tendon for irreparable rotator cuff tears.

This study presents the clinical and anatomic findings of the use of the long head of the triceps tendon for treatment of massive, irreparable rotator cuff tears. The clinical study included 19 shoulders in 18 patients with a minimum followup of 2 years who were prospectively reviewed. All patients had preoperative and postoperative functional evaluations using the UCLA scoring system. The anatomic study included 20 upper extremities that had been injected with Microfil. All patients in the clinical study were satisfied with their outcome. There was an improvement in the UCLA pain and function scores. The long head of the triceps tendon is a myotendinous vascularized transfer that is a useful reconstructive procedure in patients with massive, irreparable rotator cuff tears that continue to be symptomatic despite conventional attempts at repair.

Femoral component revision using an extensively hydroxyapatite-coated stem.

Femoral component revisions with extensively coated stems have shown promising clinical results, although concerns over stress shielding still exist. We retrospectively reviewed 59 patients undergoing femoral component revision with an extensively hydroxyapatite (HA)-coated stem. The average length of follow-up was 3.3 years (range, 2-5 years). The average preoperative Harris Hip Score was 43 points, which improved to 86 points at the latest follow-up (P < .01). The overall mechanical failure rate was 2%. No evidence of stress shielding was seen in 78% of patients. The clinical results of this series using an extensively HA-coated stem are similar to those using an extensively porous-coated stem. Long-term follow-up is required to determine if an extensively HA-coated implant will be superior to an extensively porous-coated implant with regard to stress shielding.

Intravenous mouse infection model for studying the pathology of Enterococcus faecalis infections.

An intravenous mouse infection model was used to compare the virulence of Enterococcus faecalis strains, to study bacterial localization and organ histopathology, and to examine the effects of Nramp1 and gamma interferon (IFN-gamma) on the course of infection. Infection of BALB/c mice with 5 x 10(8) CFU of E. faecalis JH2-2, MGH-2, 418, DS16C2, or OG1X revealed the following virulence ranking (from highest to lowest): MGH-2, 418, DS16C2, JH2-2, and OG1X. Discernible differences in the number of MGH-2 and JH2-2 bacteria were observed at 7 days (168 h) in the blood (P = 0.037), at 72 h in the liver (P = 0.002), and at 8 h in the spleen (P = 0.036). At these time points, the number of MGH-2 bacteria was higher in the blood and liver while the number of JH2-2 bacteria was higher in the spleen. At 72 h, livers from MGH-2-infected mice had higher numbers of coalescing aggregates of leukocytes and a greater degree of caseous necrosis than those from JH2-2-infected mice. These results indicate a correlation between the virulence of the E. faecalis strain, the number of bacteria in the liver, and the degree of histopathology of the liver at 72 h postinfection. IFN-gamma was important in E. faecalis infection, since IFN-gamma gene knockout mice had reduced mortality and massive coagulative necrosis was observed in wild-type mice. The contribution of Nramp1 was unclear, since Nramp1(-/-) mice and the respective control mice were innately resistant to E. faecalis. The mortality of mice in this model is probably due to induction of cytokine release and massive coagulative necrosis.

Experimental infection of horses with West Nile virus.

A total of 12 horses of different breeds and ages were infected with West Nile virus (WNV) via the bites of infected Aedes albopictus mosquitoes. Half the horses were infected with a viral isolate from the brain of a horse (BC787), and half were infected with an isolate from crow brain (NY99-6625); both were NY99 isolates. Postinfection, uninfected female Ae. albopictus fed on eight of the infected horses. In the first trial, Nt antibody titers reached >1:320, 1:20, 1:160, and 1:80 for horses 1 to 4, respectively. In the second trial, the seven horses with subclinical infections developed Nt antibody titers >1:10 between days 7 and 11 post infection. The highest viremia level in horses fed upon by the recipient mosquitoes was approximately 460 Vero cell PFU/mL. All mosquitoes that fed upon viremic horses were negative for the virus. Horses infected with the NY99 strain of WNV develop low viremia levels of short duration; therefore, infected horses are unlikely to serve as important amplifying hosts for WNV in nature.