Rate and factors associated with surgical site infection following aseptic revision fixation of orthopaedic trauma injuries.

PURPOSE: The primary aim of this study was to define the rate of infection following revision of fixation for aseptic failure. The secondary aims were to identify factors associated with an infection following revision and patient morbidity following deep infection. METHODS: A retrospective study was undertaken to identify patients who underwent aseptic revision surgery during a 3-year period (2017-2019). Regression analysis was used to identify independent factors associated with SSI. RESULTS: Eighty-six patients were identified that met the inclusion criteria, with a mean age of 53 (range 14-95) years and 48 (55.8%) were female. There were 15 (17%) patients with an SSI post revision surgery (n = 15/86). Ten percent (n = 9) of all revisions acquired a 'deep infection', which carried a high morbidity with a total of 23 operations, including initial revision, being undertaken for these patients as salvage procedures and three progressed to an amputation. Alcohol excess (odds ratio (OR) 1.61, 95% CI 1.01-6.36, p = 0.046) and chronic obstructive pulmonary disease (OR 11.1, 95% CI 1.00-133.3, p = 0.050) were independently associated with an increased risk of SSI. CONCLUSION: Aseptic revision surgery had a high rate of SSI (17%) and deep infection (10%). All deep infections occurred in the lower limb with the majority of these seen in ankle fractures. Alcohol excess and COPD were independent risk factors associated with an SSI and patients with a history of these should be counselled accordingly. LEVEL OF EVIDENCE: Retrospective Case Series, Level IV.

A new quinoline-based chemical probe inhibits the autophagy-related cysteine protease ATG4B.

The cysteine protease ATG4B is a key component of the autophagy machinery, acting to proteolytically prime and recycle its substrate MAP1LC3B. The roles of ATG4B in cancer and other diseases appear to be context dependent but are still not well understood. To help further explore ATG4B functions and potential therapeutic applications, we employed a chemical biology approach to identify ATG4B inhibitors. Here, we describe the discovery of 4-28, a styrylquinoline identified by a combined computational modeling, in silico screening, high content cell-based screening and biochemical assay approach. A structure-activity relationship study led to the development of a more stable and potent compound LV-320. We demonstrated that LV-320 inhibits ATG4B enzymatic activity, blocks autophagic flux in cells, and is stable, non-toxic and active in vivo. These findings suggest that LV-320 will serve as a relevant chemical tool to study the various roles of ATG4B in cancer and other contexts.

In vivo effects of two novel ALN-EP4a conjugate drugs on bone in the ovariectomized rat model for reversing postmenopausal bone loss.

UNLABELLED: Two alendronate-EP4 agonist (ALN-EP4a) conjugate drugs, C1 and C2, which differ in structure by a short linker molecule, were evaluated in ovariectomized (OVX) rats for their anabolic effects. We showed that C1 led to significant anabolic effects on cortical and trabecular bone while anabolic effects associated with C2 were minimal. INTRODUCTION: EP4as were covalently linked to ALN to create ALN-EP4a conjugate anabolic bone drugs, C1 and C2, which differ in structure by a short linker molecule in C1. When administered systemically, C1 and C2 are delivered to bone through targeted binding of ALN, where local hydrolytic enzymes liberate EP4a from ALN to exert anabolic effects. Here, we compare effects of C1 to C2 in a curative in vivo study. METHODS: Three-month-old female Sprague Dawley rats were OVX or sham operated and allowed to lose bone for 3 months. Animals were then treated via tail vein injections for 3 months and sacrificed. Treatment groups were as follows: C1L (5 mg/kg biweekly), C1H (5 mg/kg weekly), C2L (15 mg/kg monthly), C2H (15 mg/kg biweekly), OVX and sham control (phosphate-buffered saline (PBS) biweekly), and ALN/EP4a-unconjugated mixture (0.75 mg/kg each biweekly). RESULTS: MicroCT analysis showed that C1H treatment significantly increased vertebral bone mineral density (vBMD) and trabecular bone volume versus OVX controls while C2 treatments did not. Biomechanical testing showed that C1H treatment but not C2 treatments led to significant improvement in the load bearing abilities of the vertebrae compared to OVX controls. C1 stimulated endocortical bone formation and increased load bearing in femurs, while C2 did not. CONCLUSIONS: We showed that C1 led to significant anabolic effects on cortical and trabecular bone while anabolic effects associated with C2 were minimal. These results led us to hypothesize a mode of action by which presence of a linker is crucial in facilitating the anabolic effects of EP4a when dosed as a prodrug with ALN.

L-454,560, a potent and selective PDE4 inhibitor with in vivo efficacy in animal models of asthma and cognition.

Type 4 phosphodiesterases (PDE4) inhibitors are emerging therapeutics in the treatment of a number of chronic disorders including asthma, chronic obstructive pulmonary disease (COPD) and cognitive disorders. This study delineates the preclinical profile of L-454,560, which is a potent, competitive and preferential inhibitor of PDE4A, 4B, and 4D with IC50 values of 1.6, 0.5 and 1.2 nM, respectively. In contrast to the exclusive binding of cilomilast and the preferential binding of roflumilast to the PDE4 holoenzyme state (Mg2+-bound form), L-454,560 binds to both the apo-(Mg2+-free) and holoenzyme states of PDE4. The intrinsic enzyme potency for PDE4 inhibition by L-454,560 also results in an effective blockade of LPS-induced TNFalpha formation in whole blood (IC50 = 161 nM) and is comparable to the human whole blood potency of roflumilast. The cytokine profile of inhibition of L-454,560 is mainly a Th1 profile with significant inhibition of IFNgamma and no detectable inhibition of IL-13 formation up to 1 microM. L-454,560 was also found to be efficacious in two models of airway hyper-reactivity, the ovalbumin (OVA) sensitized and challenged guinea pig and the ascaris sensitized sheep model. Furthermore, L-454560 was also effective in improving performance in the delayed matching to position (DMTP) version of the Morris watermaze, at a dose removed from that associated with potential emesis. Therefore, L-454,560 is a novel PDE4 inhibitor with an overall in vivo efficacy profile at least comparable to roflumilast and clearly superior to cilomilast.

Approaching criticality in polymer-polymer systems.

The interfacial width of polyolefins blends has been probed as a function of distance away from the critical point by using neutron reflectivity. For strongly immiscible polymer pairs, the width of the interface increases slowly when the degree of immiscibility is decreased and the interfacial width varies with the interaction parameter chi of the polymers. Closer to the critical point the dependence on the degree of miscibility becomes stronger and the way in which the interfacial width diverges, as criticality is approached, is related to both the chain length and chi. The self-consistent field theory numerical calculations, with the additional contribution due to capillary waves, provides a good description of the width of the interface between two polymer bulk phases in particular at intermediate values of the degree of immiscibility.

Effects of selective prostaglandin EP4 receptor antagonist on osteoclast formation and bone resorption in vitro.

Prostaglandin estradiol (PGE(2)) stimulates bone resorption by a cyclic AMP (cAMP)-dependent mechanism that involves prostaglandin E receptors of the EP2 and EP4 subtypes. We tested a potent selective EP4 antagonist (EP4RA), which blocks PGE(2) binding to EP4 receptors. We examined the effects of EP4RA on osteoclastogenesis in murine marrow cultures, on cAMP production in primary osteoblastic (POb) cell cultures, and on bone resorption in organ cultures. EP4RA (1 micromol/L) decreased the number of tartrate-resistant acid phosphatase-positive multinucleated cells (TRAP(+) MNC) by 46%-48% in cultures treated with 0.1-1.0 micromol/L PGE(2) and by 96% in cultures treated with 0.01 micromol/L PGE(2). EP4RA also decreased TRAP(+) MNC formation by 60% in 1,25-dihydroxyvitamin D (1,25D)-treated cultures and by 62% in parathyroid hormone (PTH)-treated cultures. A chemically related analog of EP4RA that lacks antagonist activity did not inhibit TRAP(+) MNC formation. EP4RA decreased cAMP production in PGE(2)-treated POb by 44% but did not block cAMP response to PTH. EP4RA inhibited the increase in receptor activator of NF-kappaB ligand (RANKL) mRNA levels produced by PGE(2). In fetal rat long bone cultures, EP4RA decreased 45Ca release from control, unstimulated cultures by 12%-25% and from PGE(2)-stimulated cultures by 22%-37%. Because EP4RA partially inhibited osteoclastogenesis not only in response to PGE(2) but also in response to 1,25D and PTH, these results suggest that activation of the EP4 receptor may play a general role in osteoclastic bone resorption. EP4RA showed partial inhibition of PGE(2)-stimulated osteoclastogenesis at 1 micromol/L, but almost complete inhibition at 0.01 micromol/L PGE(2). This could be due to the limited efficacy of the antagonist at high concentrations of PGE(2), or an alternative pathway, such as activation of the EP2 receptor.

Stereoselective total synthesis of (+/-)-thielocin A1beta.

The stereospecific total synthesis of (+/-)-thielocin A1beta has been achieved from the common intermediate ethyl 5-formyl-2,4-dihydroxy-3,6-dimethyl benzoate (8). The racemic synthesis was achieved based on the key reaction of a 4-methyl-3,4-dihydroxy cyclohexadienone 38 with a quinone methide derived at low temperature from the fluoride ion catalyzed composition of piperidinium salt 40. The resulting condensate (31) was homologated by successive esterification with protected monomeric phenol 41 to provide, after careful removal of the protecting groups, the desired thielocin A1beta.

Etoricoxib (MK-0663): preclinical profile and comparison with other agents that selectively inhibit cyclooxygenase-2.

We report here the preclinical profile of etoricoxib (MK-0663) [5-chloro-2-(6-methylpyridin-3-yl)-3-(4-methylsulfonylphenyl) pyridine], a novel orally active agent that selectively inhibits cyclooxygenase-2 (COX-2), that has been developed for high selectivity in vitro using whole blood assays and sensitive COX-1 enzyme assays at low substrate concentration. Etoricoxib selectively inhibited COX-2 in human whole blood assays in vitro, with an IC(50) value of 1.1 +/- 0.1 microM for COX-2 (LPS-induced prostaglandin E2 synthesis), compared with an IC(50) value of 116 +/- 8 microM for COX-1 (serum thromboxane B2 generation after clotting of the blood). Using the ratio of IC(50) values (COX-1/COX-2), the selectivity ratio for the inhibition of COX-2 by etoricoxib in the human whole blood assay was 106, compared with values of 35, 30, 7.6, 7.3, 2.4, and 2.0 for rofecoxib, valdecoxib, celecoxib, nimesulide, etodolac, and meloxicam, respectively. Etoricoxib did not inhibit platelet or human recombinant COX-1 under most assay conditions (IC(50) > 100 microM). In a highly sensitive assay for COX-1 with U937 microsomes where the arachidonic acid concentration was lowered to 0.1 microM, IC(50) values of 12, 2, 0.25, and 0.05 microM were obtained for etoricoxib, rofecoxib, valdecoxib, and celecoxib, respectively. These differences in potency were in agreement with the dissociation constants (K(i)) for binding to COX-1 as estimated from an assay based on the ability of the compounds to delay the time-dependent inhibition by indomethacin. Etoricoxib was a potent inhibitor in models of carrageenan-induced paw edema (ID(50) = 0.64 mg/kg), carrageenan-induced paw hyperalgesia (ID(50) = 0.34 mg/kg), LPS-induced pyresis (ID(50) = 0.88 mg/kg), and adjuvant-induced arthritis (ID(50) = 0.6 mg/kg/day) in rats, without effects on gastrointestinal permeability up to a dose of 200 mg/kg/day for 10 days. In squirrel monkeys, etoricoxib reversed LPS-induced pyresis by 81% within 2 h of administration at a dose of 3 mg/kg and showed no effect in a fecal 51Cr excretion model of gastropathy at 100 mg/kg/day for 5 days, in contrast to lower doses of diclofenac or naproxen. In summary, etoricoxib represents a novel agent that selectively inhibits COX-2 with 106-fold selectivity in human whole blood assays in vitro and with the lowest potency of inhibition of COX-1 compared with other reported selective agents.

Synthesis, characterization, and activity of metabolites derived from the cyclooxygenase-2 inhibitor rofecoxib (MK-0966, Vioxx).

Metabolites of the COX-2 inhibitor rofecoxib (MK-0966, Vioxx) were prepared by synthetic or biosynthetic methods. Metabolites include products of oxidation, glucuronidation, reduction and hydrolytic ring opening. Based on an in vitro whole blood assay, none of the known human metabolites of rofecoxib inhibits COX-1 nor contributes significantly to the inhibition of COX-2.

Prostaglandin E2-bisphosphonate conjugates: potential agents for treatment of osteoporosis.

Conjugates of bisphosphonates (potential bone resorption inhibitors) and prostaglandin E2 (a bone formation enhancer) were prepared and evaluated for their ability to bind to bone and to liberate, enzymatically, free PGE2. The conjugate 3, an amide at C-1 of PGE2 proved to be too stable in vivo while conjugate 6, a thioester, was too labile. Several PGE2, C-15 ester-linked conjugates (18, 23, 24 and 31) were prepared and conjugate 23 was found to bind effectively to bone in vitro and in vivo and to liberate PGE2 at an acceptable rate. A 4-week study in a rat model of osteoporosis showed that 23 was better tolerated and more effective as a bone growth stimulant than daily maximum tolerated doses of free PGE2.

Quinolines as potent 5-lipoxygenase inhibitors: synthesis and biological profile of L-746,530.

Leukotriene biosynthesis inhibitors have potential as new therapeutic agents for asthma and inflammatory diseases. A series of novel substituted 2-cyanoquinolines have been synthesized and the structure activity relationships were evaluated with respect to their ability to inhibit the formation of leukotrienes via the 5-lipoxygenase enzyme. [1S,5R]-2-Cyano-4-(3-furyl)-7-¿3-fluoro-5-[3-(3 alpha-hydroxy-6,8-dioxabicyclo[3.2.1]-octanyl)]phenoxymethyl ¿quinoline (L-746,530) 3 represents a distinct class of inhibitors and possesses in vitro and in vivo potency comparable or superior to naphthalenic analog (L-739,010) 2.

2-Pyridinyl-3-(4-methylsulfonyl)phenylpyridines: selective and orally active cyclooxygenase-2 inhibitors.

A series of novel 2-pyridinyl-3-(4-methylsulfonyl)phenylpyridines has been synthesized and evaluated with respect to their ability to inhibit the isozymes of cyclooxygenase, COX-1, and COX-2. Optimum COX-2 activity is observed by introduction of a substituent at C5 of the central pyridine. 5- Chloro-3-(4-methylsulfonyl)phenyl-2-(2-methyl-5-pyridinyl)pyridine 33 was identified as the optimum compound in this series.

Substituted (pyridylmethoxy)naphthalenes as potent and orally active 5-lipoxygenase inhibitors; synthesis, biological profile, and pharmacokinetics of L-739,010.

Dioxabicyclooctanyl naphthalenenitriles have been reported as a class of potent and nonredox 5-lipoxygenase (5-LO) inhibitors. These bicyclo derivatives were shown to be metabolically more stable than their tetrahydropyranyl counterparts but were not well orally absorbed. Replacement of the phenyl ring in the naphthalenenitrile 1 by a pyridine ring leads to the potent and orally absorbed inhibitor 3g (L-739,010, 2-cyano-4-(3-furyl)-7-[[6-[3-(3-hydroxy-6,8-dioxabicyclo[3.2.1] octanyl)]-2-pyridyl]methoxy]naphthalene). Compound 3g inhibits 5-HPETE production by human 5-LO and LTB4 biosynthesis by human PMN leukocytes and human whole blood (IC50S of 20, 1.6, and 42 nM, respectively). Derivative 3g is orally active in the rat pleurisy model (inhibition of LTB4, ED50 = 0.3 mg/kg) and in the anesthetized dog model (inhibition of ex vivo whole blood LTB4 and urinary LTE4, ED50 = 0.45 and 0.23 microgram/kg/min, respectively, i.v. infusion). In addition, 3g shows excellent functional activity against ovalbumin-induced dyspnea in rats (60% inhibition at 0.5 mg/kg, 4 h pretreatment) and Ascaris-induced bronchoconstriction in conscious sheep (50% and > 85% inhibition in early and late phases, respectively at 2.5 micrograms/kg/min, i.v. infusion) and, more particularly in the conscious antigen sensitive squirrel monkey model (53% inhibition of the increase in RL and 76% in the decrease of Cdyn, at 0.1 mg/kg, po). In rats and dogs, 3g presents excellent pharmacokinetics (estimated half-lives of 5 and 16 h, respectively) and bioavailabilities (26% and 73% when dosed as its hydrochloride salt at doses of 20 and 10 mg/kg, respectively, in methocel suspension). Based on its overall biological profile, compound 3g has been selected for preclinical animal toxicity studies.

Respiratory muscle compensation for unilateral or bilateral hemidiaphragm paralysis in awake canines.

In humans and some animals, the surviving respiratory muscles are able to compensate fully for unilateral, and partially for bilateral, hemidiaphragm paralysis. To examine differential activity of individual respiratory muscles after unilateral or bilateral diaphragm paralysis, length and electromyogram (EMG) of left costal and crural diaphragm segments, parasternal intercostal, and transversus abdominis were measured directly in five awake canines after implantation with sonomicrometry transducers and bipolar EMG electrodes under three conditions: during normal breathing (NOFRZ), after infusion of local anesthetic (bupivacaine) through a cervical phrenic nerve cuff to induce reversible contralateral hemidiaphragm (CNFRZ), and after bilateral diaphragm (BIFRZ) paralysis. From NOFRZ to CNFRZ, costal, crural, parasternal, and transversus abdominis increased shortening and EMG activity to compensate for contralateral diaphragm paralysis, but the increase in activity was not equivalent for each muscle. With BIFRZ, parasternal and transversus abdominis showed further increases in activity, coordinated between both inspiration and expiration. Normalized intrabreath profiles revealed dynamic differences in development of muscle activity within each breath as paralysis worsened. Review of simultaneous muscle activities showed coordinated interactions among the compensating muscles: passive shortening of transversus, and lengthening of costal and crural, coincided with increased active inspiratory shortening of parasternal. We conclude that an integrated strategy of respiratory muscle compensation for unilateral or bilateral diaphragm paralysis occurs among chest wall, abdominal, and diaphragm segmental muscles, with relative contributions of individual muscles adjusted according to the degree of diaphragm dysfunction.

Costal and crural diaphragm function during panting in awake canines.

During natural panting for thermal regulation, the pattern of activation of the major respiratory muscles, including costal and crural diaphragm segments, is not known. We measured diaphragm segmental length, shortening, and electromyographic (EMG) activity in five chronically implanted canines awake and breathing spontaneously at rest and during a mild dry heat stress. During panting, minute ventilation increased fourfold from 5.07 l/min and respiratory rate increased from 16.9 to 192.8 breaths/min or 3.2 Hz. During panting, end-expiratory length of both costal and crural segments decreased, concurrent with significant increases in end-expiratory EMG. With the onset of panting, tidal costal shortening decreased significantly from 6.29% of end-expiratory length to 3.54%, whereas crural shortening decreased from 6.04 to 2.46%. Meanwhile, segmental EMG tended to increase during panting. During panting, intrabreath costal and crural segmental function revealed differential activation; the costal segment shortened in concert with inspiratory flow, whereas peak crural shortening occurred in expiration, almost 180 degrees out of phase with costal. The divergence in segmental shortening during panting was accompanied by a lesser shift in timing of segmental EMG. In the awake spontaneously panting canine, asynchronous costal and crural shortening may enhance gas mixing in a manner analogous to high-frequency ventilation.

Mouse monoclonal antibody to a latent epitope of leucocyte receptors for leukotriene B4.

Human blood polymorphonuclear (PMN) leucocytes and human leucocytes of the HL-60 line, which were induced to differentiate by 1,25-dihydroxyvitamin D3, express stereospecific receptors for the potent chemotactic mediator, leukotriene B4 (LTB4), that is derived by 5-lipoxygenation from arachidonic acid. Monoclonal antibodies to LTB4 receptors (LTB4-R) were generated by immunizing BALB/c mice with partially purified PMN leucocyte membrane proteins, and fusing their splenocytes with P3X63Ag8 mouse myeloma cells. Hybridoma supernatants were screened initially by binding to PMN leucocyte LTB4-R protein, which had been affinity cross-linked with aminopropylamide (APA)-LTB4 and immobilized in plastic wells through attachment of the linked APA-LTB4 to adherent Fab of monoclonal anti-LTB4. Of the three clones producing antibodies which bound to LTB4-R, 0.5 mg/ml of one IgG3k antibody, termed E2, precipitated over 90% of the [3H]LTB4-binding activity of solubilized PMN leucocyte membrane proteins. E2 also bound to a radiolabelled protein of 70,000-80,000 MW from 125I-labelled PMN leucocyte membranes [35S]-labelled HL-60 cell membranes, and PMN leucocyte membranes affinity-labelled with [3H]APA-LTB4, that was identical in size to the LTB4-R precipitated by the rabbit IgG anti-idiotypic antibodies. E2 did not bind to intact PMN leucocytes or modify the binding of [3H]LTB4 by PMN leucocytes. The binding of E2 to LTB4-R in purified membranes of PMN leucocytes was less than one-fourth of that observed for the anti-idiotypic antibodies, but increased substantially after solubilization of the LTB4-R. The E2 monoclonal antibody thus recognizes a partially latent substituent of LTB4-R, which does not contribute to combining site function.

Development of 2,3-dihydro-6-(3-phenoxypropyl)-2-(2-phenylethyl)-5-benzofuranol (L-670,630) as a potent and orally active inhibitor of 5-lipoxygenase.

Leukotrienes are potent biological mediators of allergic and inflammatory diseases and are derived from arachidonic acid through the action of the 5-lipoxygenase. In this study, the syntheses and comparative biological activities of three series of 2,3-dihydro-2,6-disubstituted-5-benzofuranols with various substituents on position 3 are described. Compounds from each series were evaluated for their ability to inhibit the production of leukotriene B4 (LTB4) in human peripheral blood polymorphonuclear (PMN) leukocytes and the 5-lipoxygenase reaction in cell-free preparations from rat PMN leukocytes. The structure-activity relationships of each series in vitro and in vivo are presented. The bioavailability, metabolism, and toxicity profile of each series are discussed. The series with no substituent at position 3 was the most potent and among the compounds in that series 2,3-dihydro-6-(3-phenoxypropyl)-2-(2-phenylethyl)-5-benzofuranol (46, L-670,630) was chosen for further development.