Successful total hip arthroplasty in a miniature horse.

OBJECTIVE: To describe the surgical treatment, postoperative management, and outcome of a miniature horse undergoing total hip arthroplasty (THA). STUDY DESIGN: Case report. ANIMALS: A 4-year-old miniature horse stallion weighing 85 kg. METHODS: The horse presented with left coxofemoral luxation of ~6 weeks duration. Computed tomography confirmed craniodorsal luxation with marked degenerative changes to the femoral head. The horse underwent THA using cementless press fit implants, including an interlocking lateral bolt for the femoral stem. RESULTS: The horse recovered well from anesthesia but suffered a coma-like episode after returning to a stable. Following treatment of presumed hypovolemia, the horse regained normal mentation and was discharged 24 days after surgery. At reassessment 12 weeks postoperatively, the horse was 2/10 left hind limb lameness at trot with good healing of the surgery site. Five months postoperatively mild (1/10) lameness remained at trot but the horse was able to canter normally on both reins. The horse has since been managed normally with no veterinary treatment required for 32 months postoperatively. CONCLUSION: Total hip arthroplasty is possible in miniature horses weighing up to 85 kg and can result in a good long-term outcome.

Assessment of intrasynovial injection in horses by contrast-enhanced ultrasonography using air bubbles created by agitation of solution.

BACKGROUND: Accuracy of intrasynovial injections can be challenging to assess in a clinical setting in horses. Contrast-enhanced ultrasonography (CEUS) using injectate agitated with air has been used to determine the success rates of synovial injections in human rheumatology. OBJECTIVES: To assess the diagnostic sensitivity and specificity of CEUS and to describe its clinical use. STUDY DESIGNS: Cadaveric study followed by a prospective descriptive observational study. METHODS: Part 1: CEUS was performed following injection of agitated methylene-blue solution targeting 13 different anatomical synovial structures from three equine cadavers. Contrast was seen as hyperechoic dots, patches or lines on ultrasonography. CEUS was classified as positive if contrast was considered to be intrasynovial and negative if contrast was considered to be extrasynovial. A second synoviocentesis was performed to determine if the injection was intrasynovial based on the presence or absence of methylene-blue. Estimates of sensitivity and specificity were calculated. Part 2: CEUS was performed following injection of agitated solutions targeting synovial structures as part of routine investigation and treatment of clinical cases. RESULTS: Part 1: CEUS was correctly classified as positive or negative in all intrasynovial and extrasynovial injections respectively. The sensitivity estimate was 100% (CI 93%-100%) and the specificity estimates was 100% (CI 16%-100%). Part 2: The technique was used safely for 26 injections (14 horses; 19 different synovial structures) administered to localise or treat lameness. Traumatic intersynovial communications or synovial membrane defects were identified using CEUS in 3 horses. MAIN LIMITATIONS: The low number of extrasynovial injections in Part 1 resulted in an imprecise specificity estimate. CONCLUSIONS: In horses, CEUS performed following intended intrasynovial injection can be useful for identifying unsuccessful injections.

Observation of direct sulfenium and selenenium group transfer from thiiranium and seleniranium ions to alkenes.

Sulfenium and selenenium ions undergo a stereospecific transfer from the corresponding three-membered ring species ("-iranium ions") to unactivated alkenes with varying facility. The thiiranium and seleniranium hexafluoroantimonates could be generated by treatment of the corresponding chloro sulfides or selenides with silver hexafluoroantimonate, followed by removal of the silver chloride by filtration. Clean (1)H, (13)C, and (77)Se NMR spectra could be recorded for these species. Treatment of the S-phenylthiiranium ion with an alkene leads to a slow transfer of the sulfenium group at 0 degrees C. However, the S-methylthiiranium ion did not transfer the sulfenium group, even at room temperature. On the other hand, both the Se-phenyl- and Se-butylseleniranium ions transferred the selenenium moiety instantaneously at -70 degrees C. By measuring the equilibrium position for these transfers from both directions, the relative stability of the 1-phenylseleniranium ions could be established: cis-tetramethylene < trans-2,3-dipropyl approximately trans-2,3-diisopropyl < cis-hexamethylene.

Inhibition of tubulin polymerization by select alkenyldiarylmethanes.

During studies on the alkenyldiarylmethane (ADAM) class of non-nucleoside reverse transcriptase inhibitors (NNRTIs), analogues were discovered that exhibit low micromolar and submicromolar cytotoxicities. Since the ADAMs are structurally related to the tubulin polymerization inhibitor CC-5079, a set of 14 ADAMs were tested for inhibition of tubulin polymerization in an attempt to identify the biological target responsible for their cytotoxicity. The results indicate that, overall, the ADAMs are poor inhibitors of tubulin polymerization. However, the two most cytotoxic compounds, 15 and 16, are in fact active as inhibitors of tubulin assembly with IC(50) values of 3.7+/-0.3 and 2.8+/-0.2 microM, respectively, and they both inhibit the binding of colchicine to tubulin. Both compounds were investigated for anticancer activity in the National Cancer Institute's panel of 60 human cancer cell lines, and both compounds consistently displayed submicromolar cytotoxicities with mean-graph midpoint (MGM) values of 0.31+/-0.08 and 0.47+/-0.09 microM, respectively.

Investigation of the alkenyldiarylmethane non-nucleoside reverse transcriptase inhibitors as potential cAMP phosphodiesterase-4B2 inhibitors.

The alkenyldiarylmethanes (ADAMs) are currently being investigated as non-nucleoside HIV-1 reverse transcriptase inhibitors (NNRTIs) of potential value in the treatment of HIV infection and AIDS. During the course of these studies, a number of ADAM analogues have been identified that protect HIV-infected cells from the cytopathic effects of the virus by an unknown, HIV-1 RT-independent mechanism. Since the phosphodiesterase 4 family is required for HIV infection, the effect of various ADAMs on the activity of PDE4B2 was investigated in an effort to determine if the ADAMs could possibly be targeting phosphodiesterases. Six compounds representative of the ADAM class were tested for inhibition of cAMP hydrolysis by PDE4B2 enzymatic activity. Four ADAMs were found to be weak inhibitors of PDE4B2 and two of them were inactive. The experimental results are consistent with an antiviral mechanism that does not include inhibition of PDE4 isoforms.

Use of a High-Throughput Phenotypic Screening Strategy to Identify Amplifiers, a Novel Pharmacological Class of Small Molecules That Exhibit Functional Synergy with Potentiators and Correctors.

Cystic fibrosis (CF) is a lethal genetic disorder caused by mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Despite recent groundbreaking approval of genotype-specific small-molecule drugs, a significant portion of CF patients still lack effective therapeutic options that address the underlying cause of the disease. Through a phenotypic high-throughput screen of approximately 54,000 small molecules, we identified a novel class of CFTR modulators called amplifiers. The identified compound, the characteristics of which are represented here by PTI-CH, selectively increases the expression of immature CFTR protein across different CFTR mutations, including F508del-CFTR, by targeting the inefficiencies of early CFTR biosynthesis. PTI-CH also augments the activity of other CFTR modulators and was found to possess novel characteristics that distinguish it from CFTR potentiator and corrector moieties. The PTI-CH-mediated increase in F508del-CFTR did not elicit cytosolic or endoplasmic reticulum-associated cellular stress responses. Based on these data, amplifiers represent a promising new class of CFTR modulators for the treatment of CF that can be used synergistically with other CFTR modulators.

Synthesis and anti-HIV activity of new metabolically stable alkenyldiarylmethane non-nucleoside reverse transcriptase inhibitors incorporating N-methoxy imidoyl halide and 1,2,4-oxadiazole systems.

The alkenyldiarylmethanes (ADAMs) are a unique class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) that are capable of inhibiting HIV-1 reverse transcriptase (RT) through an allosteric mechanism. However, the potential usefulness of the ADAMs is limited by the presence of metabolically labile methyl ester moieties that are hydrolyzed by nonspecific esterases present in blood plasma, resulting in the formation of the inactive carboxylic acid metabolites. Therefore, to discover metabolically stable ADAMs, the design and synthesis of a new class of ADAMs with N-methoxy imidoyl halide and 1,2,4-oxadiazole systems were attempted. The resulting new ADAM 6 displayed enhanced metabolic stability in rat plasma (t1/2 = 61 h) along with the ability to inhibit HIV-1 reverse transcriptase and the cytopathic effect of HIV-1RF and HIV-1IIIB at submicromolar concentrations.

Synthesis and biological evaluation of alkenyldiarylmethane HIV-1 non-nucleoside reverse transcriptase inhibitors that possess increased hydrolytic stability.

Non-nucleoside inhibitors of HIV reverse transcriptase (NNRTIs), albeit not the mainstays of HIV/AIDS treatment, have become increasingly important in highly active antiretroviral therapy (HAART) due to their unique mechanism of action. Several years ago our group identified the alkenyldiarylmethanes (ADAMs) as a potent and novel class of NNRTIs; however, the most active compounds were found to be metabolically unstable. Subsequent work has led to the synthesis of 33 analogues, with improved metabolic profiles, through the replacement of labile esters with various heterocycles, nitriles, and thioesters. As a result, a number of hydrolytically stable NNRTIs were identified with anti-HIV activity in the nanomolar concentration range. Furthermore, an improved pharmacophore model has been developed based on the new ADAM series, in which a salicylic acid-derived aryl ring is oriented cis to the side chain and the aryl ring that is trans to the side chain contains a hydrogen bond acceptor site within the plane of the ring.

A Homogeneous Cell-Based Halide-Sensitive Yellow Fluorescence Protein Assay to Identify Modulators of the Cystic Fibrosis Transmembrane Conductance Regulator Ion Channel.

Cystic fibrosis (CF), an inherited genetic disease, is caused by mutation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, which encodes an ion channel involved in hydration maintenance by anion homeostasis. Ninety percent of CF patients possess one or more copies of the F508del CFTR mutation. This mutation disrupts trafficking of the protein to the plasma membrane and diminishes function of mature CFTR. Identifying small molecule modulators of mutant CFTR activity or biosynthesis may yield new tools for discovering novel CF treatments. One strategy utilizes a 384-well, cell-based fluorescence-quenching assay, which requires extensive wash steps, but reports sensitive changes in fluorescence-quenching kinetic rates. In this study, we describe the methods of adapting the protocol to a homogeneous, miniaturized 1,536-well format and further optimization of this functional F508del CFTR assay. The assay utilizes a cystic fibrosis bronchial epithelial (CFBE41o-) cell line, which was engineered to report CFTR-mediated intracellular flux of iodide by a halide-sensitive yellow fluorescence protein (YFP) reporter. We also describe the limitations of quench rate analysis and the subsequent incorporation of a novel, kinetic data analysis modality to quickly and efficiently find active CFTR modulators. This format yields a Z' value interval of 0.61 ± 0.05. As further evidence of high-throughput screen suitability, we subsequently completed a screening campaign of >645,000 compounds, identifying 2,811 initial hits. After completing secondary and tertiary follow-up assays, we identified 187 potential CFTR modulators, which EC50's < 5 µM. Thus, the assay has integrated the advantages of a phenotypic screen with high-throughput scalability to discover new small-molecule CFTR modulators.

Crystallographic study of a novel subnanomolar inhibitor provides insight on the binding interactions of alkenyldiarylmethanes with human immunodeficiency virus-1 reverse transcriptase.

Two crystal structures have been solved for separate complexes of alkenyldiarylmethane (ADAM) nonnucleoside reverse transcriptase inhibitors (NNRTI) 3 and 4 with HIV-1 reverse transcriptase (RT). The structures reveal inhibitor binding is exclusively hydrophobic in nature and the shape of the inhibitor-bound NNRTI binding pocket is unique among other reported inhibitor-RT crystal structures. Primarily, ADAMs 3 and 4 protrude from a large gap in the back side of the binding pocket, placing portions of the inhibitors unusually close to the polymerase active site and allowing 3 to form a weak hydrogen bond with Lys223. The lack of additional stabilizing interactions, beyond the observed hydrophobic surface contacts, between 4 and RT is quite perplexing given the extreme potency of the compound (IC(50)

Synthesis and anti-HIV activity of new alkenyldiarylmethane (ADAM) non-nucleoside reverse transcriptase inhibitors (NNRTIs) incorporating benzoxazolone and benzisoxazole rings.

The HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) constitute a large and structurally diverse set of compounds, several of which are currently used in the treatment of AIDS. A series of novel alkenyldiarylmethanes (ADAMs) were designed and synthesized as part of an ongoing investigation to replace the metabolically labile methyl ester moieties found in the ADAM pharmacophore with stable modifications that retain the potent anti-HIV activity of the parent compounds. Unsurprisingly, the rat plasma half-lives of the new ADAMs were not improved when compared to the parent compounds, but all of the synthesized ADAMs inhibited the cytopathic effect of HIV-1 in cell culture. The most potent compound identified was (E)-5-[1-(3,7-dimethyl-2-oxo-2,3-dihydro-benzoxazol-5-yl)-5-methoxycarbonyl-pent-1-enyl]-2-methoxy-3-methylbenzoic acid methyl ester (7), which inhibited the cytopathic effects of both HIV-1(RF) and HIV-1(IIIB) strains in cell cultures with EC50 values of 30 and 90 nM, respectively, and inhibited HIV-1 reverse transcriptase with an IC50 of 20 nM.