Brachycephalic airway syndrome: management of post-operative respiratory complications in 248 dogs.

OBJECTIVE: As ownership of brachycephalic dog breeds rises, the surgical correction of components of brachycephalic airway syndrome (BAS) is increasingly recommended by veterinarians. This study's objective was to describe the incidence of, and strategies for the management of post-operative respiratory complications in brachycephalic dogs undergoing surgical correction of one or more components of BAS. METHODS: Medical records of 248 brachycephalic dogs treated surgically for BAS were retrospectively reviewed for demographic information, procedures performed, post-operative complications and treatment implemented, hospitalisation time, and necessity for further surgery. RESULTS: Pugs, Cavalier King Charles Spaniels and British Bulldogs were the most commonly encountered breeds. Dogs which experienced a complication were significantly older (mean was 5.5 years, compared with 4.1 years [P < 0.01]). Fifty-eight dogs (23.4%) had complications which included: dyspnoea managed with supplemental oxygen alone (7.3%, n = 18), dyspnoea requiring anaesthesia and re-intubation (8.9%, n = 22), dyspnoea necessitating treatment with a temporary tracheostomy (8.9%, n = 22), aspiration pneumonia (4%, n = 10), and respiratory or cardiac arrest (2.4%, n = 6). Five of the 22 dogs requiring anaesthesia and re-intubation deteriorated 12 or more hours after post-surgical anaesthetic recovery. The overall mortality rate in this study was 2.4% (n = 6). Age, concurrent airway pathology, and emergency presentation significantly predicted post-operative complications. CONCLUSION: Our data show the importance of close monitoring for a minimum of 24 h following surgery by an experienced veterinarian or veterinary technician. Surgical intervention for BAS symptomatic dogs should be considered at an earlier age as an elective procedure, to reduce the risk of post-operative complications.

Design and synthesis of novel alpha1a adrenoceptor-selective dihydropyridine antagonists for the treatment of benign prostatic hyperplasia.

We report the synthesis and evaluation of novel alpha1a adrenoceptor subtype-selective antagonists. Systematic modification of the lipophilic 4,4-diphenylpiperidinyl moiety of the dihydropyridine derivatives 1 and 2 provided several highly selective and potent alpha1a antagonists. From this series, we identified the 4-(methoxycarbonyl)-4-phenylpiperidine analogue SNAP 5540 (-) [(-)-63] for further characterization. When examined in an isolated human prostate tissue assay, this compound was found to have a Ki of 2.8 nM, in agreement with the cloned human receptor binding data (Ki = 2.42 nM). Further evaluation of the compound in isolated dog prostate tissue showed a Ki of 3.6 nM and confirmed it to be a potent antagonist (Kb = 1.6 nM). In vivo, this compound effectively blocked the phenylephrine-stimulated increase in intraurethral pressure (IUP) in mongrel dogs, at doses which did not significantly affect the arterial pressure (diastolic blood pressure, DBP), with a DBP Kb/IUP Kb ratio of 16. In addition, (-)-63 also showed greater than 40 000-fold selectivity over the rat L-type calcium channel and 200-fold selectivity over several G protein-coupled receptors, including histamine and serotonin subtypes. These findings prove that alpha1a adrenoceptor-subtype selective antagonists such as (-)-63 may be developed as uroselective agents for an improved treatment of BPH over nonselective alpha1 antagonists such as prazosin and terazosin, with fewer side effects.

Identification of a dihydropyridine as a potent alpha1a adrenoceptor-selective antagonist that inhibits phenylephrine-induced contraction of the human prostate.

A number of novel dihydropyridine derivatives based upon 1, 4-dihydro-3-(methoxycarbonyl)-2, 6-dimethyl-4-(4-nitrophenyl)-5-((3-(4, 4-diphenylpiperidin-1-yl)propyl)aminocarbonyl)pyridine (4) have been synthesized and tested at cloned human alpha adrenoceptors as well as the rat L-type calcium channel. Within this compound series, 5-(aminocarbonyl)-1,4-dihydro-2, 6-dimethyl-4-(4-nitrophenyl)-3-((3-(4, 4-diphenylpiperidin-1-yl)propyl)aminocarbonyl)pyridine (19) displayed good binding affinity and selectivity for the alpha1a adrenoceptor (pKi = 8.73) and potently inhibited (pA2 = 9.23) phenylephrine-induced contraction of the human prostate.

Design and synthesis of novel alpha(1)(a) adrenoceptor-selective antagonists. 1. Structure-activity relationship in dihydropyrimidinones.

Dihydropyrimidinones such as compound 12 exhibited high binding affinity and subtype selectivity for the cloned human alpha(1a) receptor. Systematic modifications of 12 led to identification of highly potent and subtype-selective compounds such as (+)-30 and (+)-103, with high binding affinity (K(i) = 0.2 nM) for alpha(1a) receptor and greater than 1500-fold selectivity over alpha(1b) and alpha(1d) adrenoceptors. The compounds were found to be functional antagonists in human, rat, and dog prostate tissues. Compound (+)-103 exhibited excellent selectively to inhibit intraurethral pressure (IUP) as compared to lowering diastolic blood pressure (DBP) in mongrel dogs (K(b)(DBP)/K(b)(IUP) = 40) suggesting uroselectivity for alpha(1a)-selective compounds.

Design and synthesis of novel alpha(1)(a) adrenoceptor-selective antagonists. 2. Approaches to eliminate opioid agonist metabolites via modification of linker and 4-methoxycarbonyl-4-phenylpiperidine moiety.

We have previously described compound 1a as a high-affinity subtype selective alpha(1a) antagonist. In vitro and in vivo evaluation of compound 1a showed its major metabolite to be a mu-opioid agonist, 4-methoxycarbonyl-4-phenylpiperidine (3). Several dihydropyrimidinone analogues were synthesized with the goal of either minimizing the formation of 3 by modification of the linker or finding alternative piperidine moieties which when cleaved as a consequence of metabolism would not give rise to mu-opioid activity. Modification of the linker gave several compounds with good alpha(1a) binding affinity (K(i) = < 1 nM) and selectivity (>300-fold over alpha(1b) and alpha(1d)). In vitro analysis in the microsomal assay revealed these modifications did not significantly affect N-dealkylation and the formation of the piperidine 3. The second approach, however, yielded several piperidine replacements for 3, which did not show significant mu-opioid activity. Several of these compounds maintained good affinity at the alpha(1a) adrenoceptor and selectivity over alpha(1b) and alpha(1d). For example, the piperidine fragments of (+)-73 and (+)-83, viz. 4-cyano-4-phenylpiperidine and 4-methyl-4-phenylpiperidine, were essentially inactive at the mu-opioid receptor (IC(50) > 30 microM vs 3 microM for 3). Compounds (+)-73 and (+)-83 were subjected to detailed in vitro and in vivo characterization. Both these compounds, in addition to their excellent selectivity (>880-fold) over alpha(1b) and alpha(1d), also showed good selectivity over several other recombinant human G-protein coupled receptors. Compounds (+)-73 and (+)-83 showed good functional potency in isolated human prostate tissues, with K(b)s comparable to their in vitro alpha(1a) binding data. In addition, compound (+)-73 also exhibited good uroselectivity (DBP K(b)/IUP K(b) > 20-fold) in the in vivo experiments in dogs, similar to 1a.

Design and synthesis of novel alpha(1)(a) adrenoceptor-selective antagonists. 4. Structure-activity relationship in the dihydropyrimidine series.

We have previously disclosed dihydropyridines such as 1a,b as selective alpha(1a) antagonists as a potential treatment for benign prostatic hyperplasia (BPH). The propensity of dihydropyridines toward an oxidation led us to find suitable replacements of the core unit. The accompanying papers describe the structure-activity relationship (SAR) of dihydropyrimidinones 2a,b as selective alpha(1a) antagonists. We report herein the SAR of dihydropyrimidines such as 4 and highlight the similarities and differences between the dihydropyrimidine and dihydropyrimidinone series of compounds.

Characterization of specific binding of [125I]L-762,459, a selective alpha1A-adrenoceptor radioligand to rat and human tissues.

L-762,459 ((+/-)1-(3-¿[5-carbamoyl-2-2-[(4-hydroxy-3-iodobenzimidoyl)-amino] -ethoxy-methy¿-6-methyl-4-(4-nitropheny)-1,4-dihydropyridine -3-carbonyl]-amino¿-propyl)-4-phenyl-1-piperidine-4-carboxylic acid methyl ester), an analog of a series of dihydropyridines previously reported to be selective alpha1A-adrenoceptor subtype antagonists was found to have alpha1A-adrenoceptor subtype selectivity (Ki (nM), la = 1.3, lb = 240, Id = 280). Specific [125I]L-762,459 binding was detected in rat cerebral cortex, hippocampus, vas deferens, kidney, heart and prostate tissues known to contain the alpha1A-adrenoceptor subtype, but not in tissues known to contain alpha1B-adrenoceptor (spleen, liver) and alpha1D-adrenoceptor (aorta). Scatchard analysis of [125I]L-762,459 binding in rat cerebral cortex and prostate indicated a single binding site with a Kd of 0.7 nM and Bmax of 11 (cerebral cortex) and 1 (prostate) pmole/g tissue. Specific and saturable [125I]L-762,459 binding was also found in human cerebral cortex, liver, prostate and vas deferens (Kd = 0.2-0.4 nM, Bmax = 0.4-4 pmole/g tissue). The specific binding in rat and human tissues was competed by non-selective alpha1-adrenoceptor compounds (Ki values in nM: prazosin (0.14-1.2), terazosin (1.8-5.9) and phentolamine (2.4-11)) and selective alpha1A-adrenoceptor compounds [Ki values in nM: (+) niguldipine (0.04-1.2) and SNAP 5399 ((+/-)-2-((2-aminoethyl)oxy)methyl-5-carboxamido-6-ethyl-4-(4-nitropheny l)-3-N-(3-(4,4-diphenylpiperidin-1-yl)propyl)carboxamido-1,4-dihyd ropyridine hydrate (0.5-4.8)]. The results were consistent with the selective binding of [125I]L-762,459 to the alpha1A-adrenoceptor. The specific labeling of the alpha1A-adrenoceptor subtype by [125I]L-762,459 may make it a useful tool to localize the distribution of the alpha1A-adrenoceptor.

Warfarin pharmacokinetics in the horse.

The pharmacokinetics of racemic warfarin were studied in 6 adult horses. After IV administration, the plasma concentration of warfarin showed a biphasic decline in time. Analysis of the data, according to 2-compartment kinetics, revealed the following constants: biological half-life was 13.3 hours, apparent volume of distribution was 0.46 L X kg-1, body clearance was 25.3 ml X hour-1 X kg-1. Warfarin was bound (91.5%) to plasma proteins. Unchanged warfarin was not detected in the urine. Absorption from the gastrointestinal tract was almost complete. Concentrations of warfarin in tissue were examined in 4 foals that were given 1 g of warfarin orally 14 hours before they were euthanatized. The concentrations in the kidney were higher than those in plasma (about 2 fold). The hypothrombogenic effect of the acute IV and oral administration of warfarin (0.75 mg/kg of body weight) was noticeable only after 60 hours and lasted for about 30 hours. The effect was weak; the maximum effect was a prolongation of 2.3 s (from 11.5 s to 13.8 s) in thromboplastin time.

Determination of the relative and absolute stereochemistry of a potent and alpha1A-selective adrenoceptor antagonist.

The binding affinities and selectivities of antagonists 1-4 for the alpha1A-adrenoceptor are dependent on the stereochemical orientation of the groups at the C-4 and C-5 positions of the oxazolidinone ring. The unambiguous assignment of the relative and absolute configurations of the diastereomers of SNAP 7915 (1) is reported.

Localization of the alpha 1A-adrenoceptor in the human prostate.

PURPOSE: We determined the tissue localization of the alpha 1a-adrenoceptor in the human prostate. MATERIALS AND METHODS: Autoradiographic localization of the alpha 1a-adrenoceptor in the human prostate was determined by performing competitive displacement experiments on slide mounted tissue sections using the ligand 125iodine-2-(-[4-hydroxyphenyl]-ethyl-aminomethyl)tetralone (125I-Heat), and the alpha 1-antagonists WB-4101 (4 x 10(-8) M.) and 5-carboxamido-2,6-diethyl-1,4-dihydro-3-[N-(3-[4-hydroxy-4-phenylpipe ridin- yl]propyl)]carboxamido-4-(4-nitrophenyl) (SNAP 5272, 3 x 10(-7) M.). Under these experimental conditions, WB-4101 and SNAP 5272 are selective alpha 1a/alpha 1d-adrenoceptor and alpha 1a-adrenoceptor antagonists, respectively. The autoradiographs were quantitatively analyzed using a computer image analysis system. RESULTS: Specific 125I-Heat binding associated with the epithelium and stroma were independently analyzed. WB-4101 and SNAP 5272 inhibited 100% of the specific 125I-Heat binding in the stroma, suggesting that all of the stromal alpha 1-adrenoceptors are of the alpha 1a subtype. WB-4101 inhibited none of the specific 125I-Heat binding in the epithelium, suggesting that the alpha 1-adrenoceptor in the epithelium is of the alpha 1b subtype. SNAP 5272 displaced only 25% of the specific 125I-Heat binding in the epithelium, suggesting that a relatively small percentage of the epithelial alpha 1-adrenoceptor is of the alpha 1a subtype. CONCLUSIONS: To our knowledge, our study represents the first cellular localization of the alpha 1-adrenoceptor subtypes in the human prostate using highly selective alpha 1-adrenoceptor antagonists and is consistent with the physiological observation that the activity of prostatic smooth muscle is mediated by the alpha 1a-adrenoceptor.

Diversity in steroidogenesis of symbiotic microorganisms from planthoppers.

Ergosta-5,7,24(28)-trien-3ß-ol (trienol)6 was isolated from the intracellular symbiotes (symbiotic microorganisms) of the planthoppers,Nilaparvata lugens andLaodelphax striatellus. The steroidogenic end product of the symbiotes was found to depend on the environmental conditions, i.e., although trienol6 was produced under symbiotic conditions, ergosterol4 was formed in aerobic culture. When the normal diet was replaced by a steroiddeficient artificial one, the ratio of 24-methylenecholesterol5 to total insect sterols was significantly increased. The above study offers further corroborating evidence for our assumption that the host insects require 24-methylenecholesterol5 as an alternate source for cholesterol1 under certain environmental conditions. In our previous biotransformation experiments, 24methylenecholesterol5 was shown to be an immediate precursor of cholesterol1. However, the complexity of the insects' vital functions hindered all subsequent attempted transformations into cholesterol1 of sitosterol2, trienol6, and ergosterol4, under injection conditions.

Long-term sub-antimicrobial doxycycline (Periostat) as adjunctive management in adult periodontitis: effects on subgingival bacterial population dynamics.

Previous trials had indicated that various schedules of sub-antimicrobial doxycycline significantly reduced gingival crevicular fluid (GCF) collagenase activity in adult patients with periodontitis with no evidence of emergent tetracycline-resistant (Tcr) marker oral flora. The purpose of this nine-month study was to expand these observations, emphasizing newer microbial diagnostic methods. Subgingival paper point samples were obtained at baseline (BL), 3, 6, and 9 months. Four subject treatment groups in a double-blind design were evaluated by mechanical scaling and root planing (SRP) and/or 20 mg doxycycline BID (Periostat). Thirty-eight patients entered the study at baseline (BL). Dark-field microscopy on 260 samples showed that morphotype distribution was independent of treatment schedule. Culture analysis of the 3 most prevalent isolates recovered showed that Streptococcus and Prevotella species accounted for approximately 85% of the 724 cultures. There did not appear to be any overgrowth or replacement by opportunistic oral flora. Of 658 susceptibility patterns evaluated by Etest, the MIC50/90 and mode MIC showed stable patterns, independent of treatment group. Our findings were different from those of previously published reports, but may be partly explained by the lack of universally standardized methods in oral microbiology and interpretive criteria for susceptibility testing.

Localization of mRNA and receptor binding sites for the alpha 1a-adrenoceptor subtype in the rat, monkey and human urinary bladder and prostate.

PURPOSE: To localize the mRNAs and receptor binding sites for the alpha 1a/A, alpha 1b/B and alpha 1d/D- adrenoceptor (AR) subtypes in the rat, monkey and human urinary bladder and prostate. MATERIALS AND METHODS: alpha 1-AR mRNAs were localized on slide mounted tissue sections by in situ hybridization using [35S]-labeled subtype specific oligonucleotide probes. alpha 1-AR receptor binding sites were localized on slide mounted tissue sections by competitive displacement of [3H]-prazosin using subtype selective ligands. RESULTS: Only the alpha 1a-AR subtype mRNA was discernible by in situ hybridization. The alpha 1a-AR mRNA was localized in all smooth muscle areas of the rat, monkey and human urinary bladder and prostate. High levels of alpha 1a mRNA were detected in bladder dome and bladder base urothelium. Competitive displacement studies using the alpha 1A-AR selective ligand SNAP 5272 revealed that the alpha 1A-AR represented over 80% of the total alpha 1-AR in monkey bladder and prostate. In general, localization of the alpha 1A-AR corresponded to the alpha 1a-AR mRNA localization, that is, receptor protein was localized to smooth muscle areas of the bladder dome, trigone and base and prostate. One notable exception was the bladder urothelium, which contained high levels of alpha 1a-AR mRNA, but undetectable levels of alpha 1A-AR protein. The alpha 1a-AR mRNA appeared to be transcribed but not translated in bladder urothelium. CONCLUSIONS: The alpha 1A-AR represents the major subtype in the smooth muscle of rat, monkey and human urinary systems. Selective alpha 1A-AR agents are therefore potentially useful in the treatment of multiple urinary smooth muscle related disorders.

The alpha 1-adrenergic receptor that mediates smooth muscle contraction in human prostate has the pharmacological properties of the cloned human alpha 1c subtype.

Molecular cloning studies have revealed the existence of three subtypes of alpha 1-adrenergic receptors. However, the link between any individual subtype and its functional role in the body has remained elusive. In an effort to bridge the gap between molecular biology and pathophysiology, we have chosen a model smooth muscle system, the human prostate, and investigated the role of alpha 1 subtypes in this tissue. To determine which alpha 1-adrenergic receptor subtype mediates the contractile response of the human prostate, we first studied the pharmacological properties of three cloned human alpha 1 subtypes (alpha 1a/d, alpha 1b, and alpha 1c). Prazosin, terazosin, doxazosin, alfuzosin, and abanoquil showed no selectivity for the human alpha 1 subtypes. WB-4101 and 5-methylurapidil showed a rank order of potency of alpha 1c > alpha 1a/d >> alpha 1b. Indoramin and (+)-niguldipine were selective for the alpha 1c-adrenergic receptor, with at least 10-fold lower affinity at either alpha 1a/d or alpha 1b subtypes. SK&F104856 was found to be 6-fold more potent at the alpha 1a/d receptor subtype than at alpha 1b- or alpha 1c-adrenergic receptors. We next determined the potency of these antagonists to inhibit the phenylephrine-induced contraction of human prostatic tissue in vitro. The potencies of indoramin, 5-methylurapidil, and SK&F104856 to inhibit the contractile response and to displace [3H]prazosin from the cloned human alpha 1c subtype were similar. Our data suggest that the alpha 1 receptor that mediates the contraction of human prostate smooth muscle has the pharmacological properties of the cloned human alpha 1c-adrenergic receptor. The findings of the present study suggest that selective alpha 1c-adrenergic receptor antagonists may be clinically more efficacious and better tolerated agents for the treatment of symptomatic benign prostatic hyperplasia.

Design and synthesis of novel dihydropyridine alpha-1a antagonists.

A series of analogs of SNAP 5150 containing heteroatoms at C2 or C6 positions is described. Herein, we report that the presence of alkyl substituted heteroatoms at the C2(6)-positions of the dihydropyridine are well tolerated. In addition, 15 inhibited the phenylephrine induced contraction of dog prostate tissue with a Kb of 1.5 nM and showed a Kb (DBP, dogs, microg/kg)/Kb (IUP, dogs, microg/kg) ratio of 14.8/2.5.

Modeling and mutagenesis of the human alpha 1a-adrenoceptor: orientation and function of transmembrane helix V sidechains.

A 3-dimensional model of the seven transmembrane helical segments (TMs) of the human alpha 1a-adrenoceptor was initially built by analogy to the known structure of bacteriorhodopsin. However, the rotational orientation of TM V about its helical axis, and the roles of several TM V residues in ligand binding and receptor activation remained in question. Accordingly, we determined the effects of six site-specific mutations in TM V on binding affinity and functional potency of a structurally diverse series of agonists and antagonists. Mutation of Ser 192 and Phe 193 disrupted the binding of many of the tested ligands, as measured by displacement of [3H]prazosin. In addition, mutation of Ser 188, Ser 192, and Phe 193 disrupted receptor activation, as measured by [3H]inositol phosphate formation. On the basis of these results, a specific rotational orientation of TM V is proposed as part of a revised receptor model, which also takes into account more recently reported information about the structure of rhodopsin. This revised alpha 1a-adrenoceptor model accounts for direct interactions which are proposed between Ser 188 and Ser 192 and the meta and para hydroxyl groups of norepinephrine, respectively, in the G-protein coupled receptor state.

Identification and characterization of two G protein-coupled receptors for neuropeptide FF.

The central nervous system octapeptide, neuropeptide FF (NPFF), is believed to play a role in pain modulation and opiate tolerance. Two G protein-coupled receptors, NPFF1 and NPFF2, were isolated from human and rat central nervous system tissues. NPFF specifically bound to NPFF1 (K(d) = 1.13 nm) and NPFF2 (K(d) = 0.37 nm), and both receptors were activated by NPFF in a variety of heterologous expression systems. The localization of mRNA and binding sites of these receptors in the dorsal horn of the spinal cord, the lateral hypothalamus, the spinal trigeminal nuclei, and the thalamic nuclei supports a role for NPFF in pain modulation. Among the receptors with the highest amino acid sequence homology to NPFF1 and NPFF2 are members of the orexin, NPY, and cholecystokinin families, which have been implicated in feeding. These similarities together with the finding that BIBP3226, an anorexigenic Y1 receptor ligand, also binds to NPFF1 suggest a potential role for NPFF1 in feeding. The identification of NPFF1 and NPFF2 will help delineate their roles in these and other physiological functions.