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.

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.

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.

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.

Development of enzyme-linked immunosorbent assays for measurement of leukotrienes and prostaglandins.

Enzyme-linked immunosorbent assays (ELISAs) were developed for the leukotrienes LTC4 and LTB4 and the prostaglandins 6-keto PGF1 alpha and thromboxane (TxB2). In an indirect assay procedure for all 4 eicosanoids a BSA conjugate of the leukotrienes or an ovalbumin conjugate of the prostaglandins was absorbed to polystyrene microtiter plates. Samples containing the respective eicosanoids were incubated in the coated wells with specific rabbit antisera. The wells were then incubated successively with a goat anti-rabbit antibody linked to fluorescein and a rabbit anti-fluorescein antibody linked to alkaline phosphatase. The resultant assays for LTC4, LTB4, 6-keto PGF1 alpha, and TxB2, gave steep, sensitive inhibition curves; IC50s were 0.2, 10, 1, and 0.4 pmol respectively with minimal cross-reactivity to other eicosanoids. The sensitivities and specificities were comparable to those found in the RIA, and the levels determined in this assay correlate well with those determined in non-immunological assays.

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.

Preparation and analgesic properties of amino acid derivatives of (-)-5,9 alpha-diethyl-2'-hydroxybenzomorphan.

The N-arginyl derivative of methionine-enkephalin (fragment 60-65 of beta-lipotropin) has been shown to be equiactive with the parent pentapeptide, despite the fact that the tyrosine amino group in this compound has been neutralized by the formation of an amide linkage. A series of N-(amino acid) derivatives of (-)-5,9 alpha-diethyl-2'-hydroxybenzomorphan was prepared and evaluated for analgesic activity. In vitro activities were found to vary greatly, depending on the nature of the amino acid used. The N-arginyl derivative was found to be equipotent to (-)-5,9 alpha-diethyl-2'hydroxybenzomorphan and also to methionine-enkephaline in the naloxone binding assay.

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.

Stereoselective determination of (beta S,gamma R and beta R,gamma S)-4-[3-(4-acetyl-3-hydroxy-2-propylphenoxy) propylthio]-gamma-hydroxy-beta-methylbenzenebutanoic acid in human and rat plasma by normal-phase high-performance liquid chromatography.

A stereoselective high-performance liquid chromatographic method was developed for the determination of the enantiomeric composition comprising (beta S,gamma R and beta R,gamma S)-4-[3-(4-acetyl-3-hydroxy-2-propylphenoxy) propylthio]-gamma-hydroxy-beta-methylbenzenebutanoic acid in human and rat plasma. Both enantiomers, (beta S,gamma R)- and (beta R,gamma S)-1, were isolated from the plasma by liquid-liquid extraction, the organic phase was evaporated to dryness, and the residue was lactonized with p-toluenesulfonic acid followed by derivatization with R(+)-alpha-methylbenzylamine to form diastereomeric Schiff base adducts. Separation was performed on a silica column (Supelcosil) with a mobile phase composed of 97.8% hexane, 2% isopropyl alcohol, and 0.2% triethylamine. The column eluant was monitored with a variable wavelength UV detector set at 280 nm (0.005 AUFS). Based on the peak area ratios, the method shows excellent linear relationships with their corresponding concentrations and satisfactory intraday and interday assay precision and accuracy. The detection limit of this method is 25 ng/mL in plasma for each enantiomer. The method has been applied to the assay of plasma obtained from human subjects administered 1 in safety and tolerability studies and from rats administered 1 intravenously or per os.

Development of novel leukotriene--based anti-asthma drugs: MK-886 and MK-571.

A potent, selective and orally active receptor antagonist of leukotriene D4, MK-571, was discovered and developed from a styrylquinoline lead structure based on a hypothetical model of the leukotriene D4 receptor. MK-571 blocks the action of LTD4 in animals and man, and is effective in a number of animal models of antigen-induced bronchoconstriction at plasma concentration at or below 2 micrograms/mL. MK-571 also blocks antigen-induced asthmatic responses in man. In addition a series of 2-indolealkanoic acids was discovered to be inhibitors of leukotriene biosynthesis. From this series, MK-886, a nanomolar inhibitor of leukotriene biosynthesis was developed. The mechanism of action of MK-886 has been found to be the inhibition of activation of the 5-lipoxygenase enzyme. This inhibition is mediated by interaction with a specific 18 kD protein termed 5-lipoxygenase activating protein (FLAP). MK-886 is an inhibitor of leukotriene biosynthesis and of antigen-induced bronchoconstriction in animal models and in asthmatic men.

L-648,051, a potent and specific aerosol active leukotriene D4 antagonist.

L-648,051 (I) is a potent, competitive and selective antagonist of the leukotriene D4 receptor in guinea pig and human lung tissue. Its activity on isolated smooth muscle preparations and its superiority relative to other agents (L-649,923 (II) or FPL 55712) in reversing ongoing contraction to LTD4 was somewhat unexpected in light of the results of receptor binding studies (Ki = 6.2 microM for L-648,051 versus 0.4 microM for L-649,923 and 2.0 microM for FPL 55712 on guinea pig lung membranes). The reasons for this observed superiority are unknown but may relate to the rate at which various antagonists equilibrate with the leukotriene receptors on various tissues. The physical properties (polarity) of L-648,051 may contribute to this enhanced rate of equilibration. The rapid metabolism and elimination of L-648,051 in vivo should be an advantage for a topical agent by minimizing systemic exposure. The potential role for L-648,051 as a novel aerosol therapy for asthma is now being investigated.

Multichannel electrochemical microbial detection unit.

A compact multichannel unit for electrochemical detection of microorganisms that automatically displays detection time length is described. This unit was successfully tested with various members of the Enterobacteriaceae group.