Performance and reading time of lung nodule identification on multidetector CT with or without an artificial intelligence-powered computer-aided detection system.

AIM: To compare the performance and reading time of different readers using automatic artificial intelligence (AI)-powered computer-aided detection (CAD) to detect lung nodules in different reading modes. MATERIALS AND METHODS: One hundred and fifty multidetector computed tomography (CT) datasets containing 340 nodules ≤10 mm in diameter were collected retrospectively. A CAD with vessel-suppressed function was used to interpret the images. Three junior and three senior readers were assigned to read (1) CT images without CAD, (2) second-read using CAD in which CAD was applied only after initial unassisted assessment, and (3) a concurrent read with CAD in which CAD was applied at the start of assessment. Diagnostic performances and reading times were compared using analysis of variance. RESULTS: For all readers, the mean sensitivity improved from 64% (95% confidence interval [CI]: 62%, 66%) for the without-CAD mode to 82% (95% CI: 80%, 84%) for the second-reading mode and to 80% (95% CI: 79%, 82%) for the concurrent-reading mode (p<0.001). There was no significant difference between the two modes in terms of the mean sensitivity, specificity, and area under the receiver operating characteristic curve (AUC) for both junior and senior readers and all readers (p>0.05). The reading time of all readers was significantly shorter for the concurrent-reading mode (124 ± 25 seconds) compared to without CAD (156 ± 34 seconds; p<0.001) and the second-reading mode (197 ± 46 seconds; p<0.001). CONCLUSION: In CAD for lung nodules at CT, the second-reading mode and concurrent-reading mode may improve detection performance for all readers in both screening and clinical routine practice. Concurrent use of CAD is more efficient for both junior and senior readers.

Fusion of microlitre water-in-oil droplets for simple, fast and green chemical assays.

A simple format for microscale chemical assays is proposed. It does not require the use of test tubes, microchips or microtiter plates. Microlitre-range (ca. 0.7-5.0 µL) aqueous droplets are generated by a commercial micropipette in a non-polar matrix inside a Petri dish. When two droplets are pipetted nearby, they spontaneously coalesce within seconds, priming a chemical reaction. Detection of the reaction product is accomplished by colorimetry, spectrophotometry, or fluorimetry using simple light-emitting diode (LED) arrays as the sources of monochromatic light, while chemiluminescence detection of the analytes present in single droplets is conducted in the dark. A smartphone camera is used as the detector. The limits of detection obtained for the developed in-droplet assays are estimated to be: 1.4 nmol (potassium permanganate by colorimetry), 1.4 pmol (fluorescein by fluorimetry), and 580 fmol (sodium hypochlorite by chemiluminescence detection). The format has successfully been used to monitor the progress of chemical and biochemical reactions over time with sub-second resolution. A semi-quantitative analysis of ascorbic acid using Tillman's reagent is presented. A few tens of individual droplets can be scanned in parallel. Rapid switching of the LED light sources with different wavelengths enables a spectral analysis of multiple droplets. Very little solid waste is produced. The assay matrix is readily recycled, thus the volume of liquid waste produced each time is also very small (typically, 1-10 µL per analysis). Various water-immiscible translucent liquids can be used as the reaction matrix: including silicone oil, 1-octanol as well as soybean cooking oil.

Characterization and overexpression of a novel ß-agarase from Thalassomonas agarivorans.

AIMS: The agarase from Thalassomonas agarivorans BCRC 17492 was cloned and overexpressed in Escherichia coli. The characterization of the novel agarase was performed. METHODS AND RESULTS: The genomic library of T. agarivorans BCRC 17492 was constructed for screening agarase gene. The novel ß-agarase, namely AgaB1, was successfully identified and shared only 57% identity to reported agarase from Alteromonas sp. To characterize the AgaB1 protein, the recombinant AgaB1 can be obtained by heterologous expression in E. coli. The agarase activity of AgaB1 was achieved at 30·25 U per mg at 35°C. According to the analysis of optimal conditions, the highest activity of AgaB1 was attained at 40°C, pH 7·4 and 200 mmol l(-1) NaCl, and half-life of AgaB1 can be maintained for almost 1 h at 40°C. Further determination of substrate hydrolysis indicated that AgaB1 had possession of both endo- and exolytic activity, and neoagarobiose was the major hydrolysis product by TLC and high-performance liquid chromatograph/mass spectrometer (HPLC/MS) analysis. CONCLUSIONS: We have successfully cloned and overexpressed the novel ß-agarase from T. agarivorans BCRC 17492 in E. coli. The high yield and detailed characterization of recombinant AgaB1 was provided. SIGNIFICANT AND IMPACT OF THE STUDY: AgaB1 was the first ß-agarase that was cloned and described from Thalassomonas species. In the light of properties of AgaB1, it has the potential as the biocatalyst for industrial applications.

Contact behavior of focused ion beam deposited Pt on p-type Si nanowires.

Pt contact on p-Si nanowires (NWs) using Ga-ion-induced deposition by a focused ion beam was formed with a specific contact resistance (rho(c)) of 1.54 x 10(-6) Omega cm(2). Ohmic behavior is caused by Ga-ion-induced amorphization of Si NWs underneath the Pt contact. A very low Schottky barrier height associated with interface states raised from Pt-amorphized Si junction and with an image force induced by the applied bias can be implemented to elucidate ultralow rho(c). The value of rho(c) lower than that of any known contact to Si NWs demonstrates a practical method for integrating NWs in devices and circuits.

Species and gender differences in the formation of an active metabolite of a substituted 2,4-thiazolidinedione insulin sensitizer.

1. The metabolism of a substituted 2,4-thiazolidinedione (P1) with dual PPARalpha/gamma activity was evaluated in male and female rats, dogs and monkeys. A para-hydroxylated metabolite (M1) with potent PPARgamma-selective agonist, was a major circulating drug-related component in female rats, dogs and monkeys, but not in male rats (M1-to-P1 exposure ratio of <1, 3-5, 5 and 5-11 in male rat, monkey, female rat, and dog, respectively). 2. M1 (%) formed in vitro (5, 53, 57-65, 67 and 67% in male rat, monkey, female rat, dog, and human liver microsomes, respectively), rank ordered with M1 (%) formed in vivo (24-45, 53-57, 78, 75-85%, for male rat, monkey, female rat and dog, respectively, after oral administration of P1). 3. The plasma clearance of M1 was higher in male rats (32 ml min(-1) kg(-1) compared with 6, 7 and 2 ml min(-1) kg(-1) in female rat, male monkey and male dogs, respectively). 4. The low amounts of M1 observed in male rats, with the appearance of products of the cleavage of the propyl group between the phenyl groups was probably due to the presence of the sex-specific CYP2C11, which cleaves P1 at the propyl bridge. None of the CYPs present in female rats cleaved P1 at this site and M1 was only produced by CYP2C6. In humans, only CYP2C8 and the polymorphic CYP2C19 produced M1.

Parallel extraction columns and parallel analytical columns coupled with liquid chromatography/tandem mass spectrometry for on-line simultaneous quantification of a drug candidate and its six metabolites in dog plasma.

A method with parallel extraction columns and parallel analytical columns (PEC-PAC) for on-line high-flow liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed and validated for simultaneous quantification of a drug candidate and its six metabolites in dog plasma. Two on-line extraction columns were used in parallel for sample extraction and two analytical columns were used in parallel for separation and analysis. The plasma samples, after addition of an internal standard solution, were directly injected onto the PEC-PAC system for purification and analysis. This method allowed the use of one of the extraction columns for analyte purification while the other was being equilibrated. Similarly, one of the analytical columns was employed to separate the analytes while the other was undergoing equilibration. Therefore, the time needed for re-conditioning both extraction and analytical columns was not added to the total analysis time, which resulted in a shorter run time and higher throughput. Moreover, the on-line column extraction LC/MS/MS method made it possible to extract and analyze all seven analytes simultaneously with good precision and accuracy despite their chemical class diversity that included primary, secondary and tertiary amines, an alcohol, an aldehyde and a carboxylic acid. The method was validated with the standard curve ranging from 5.00 to 5000 ng/mL. The intra- and inter-day precision was no more than 8% CV and the assay accuracy was between 95 and 107%.

Mycobacterium immunogenum sp. nov., a novel species related to Mycobacterium abscessus and associated with clinical disease, pseudo-outbreaks and contaminated metalworking fluids: an international cooperative study on mycobacterial taxonomy.

PCR-restriction enzyme pattern analysis of a 439 bp hsp65 gene segment identified 113 unique isolates among non-pigmented rapidly growing mycobacteria (RGM) from clinical and environmental sources that failed to match currently recognized species patterns. This group represented 40% of isolates recovered from bronchoscope contamination pseudo-outbreaks, 0% of disease-associated nosocomial outbreaks and 4% of routine clinical isolates of the Mycobacterium abscessus/Mycobacterium chelonae group submitted to the Mycobacteria/Nocardia laboratory for identification. It is grouped within the Mycobacterium fortuitum complex, with growth in less than 7 d, absence of pigmentation, positive 3-d arylsulfatase reaction and growth on MacConkey agar without crystal violet. It exhibited overlapping biochemical, antimicrobial susceptibility and HPLC characteristics of M. abscessus and M. chelonae. By 16S rRNA gene sequencing, these isolates comprised a homogeneous group with a unique hypervariable region A sequence and differed by 8 and 10 bp, respectively, from M. abscessus and M. chelonae. Surprisingly, this taxon contained two copies of the ribosomal operon, compared with single copies in the two related species. By DNA-DNA hybridization, this new group exhibited <30% homology with recognized RGM species. The name Mycobacterium immunogenum sp. nov. is proposed for this new taxon.

Use of on-line hydrogen/deuterium exchange to facilitate metabolite identification.

Biotransformation studies performed on an investigational compound (I, represented by R1-CH(NH(2))-CO-N(R2)-CH(2)-S-R3) led to the identification of five metabolites (M1-M5). Based on LC/MS (liquid chromatography/mass spectrometry) analysis which included the use of H(2)O and D(2)O in the mobile phases, they were identified as the sulfoxide (M1), sulfone (M2), carbamoyl glucuronide (M3), N-glucuronide (M4), and N-glucoside (M5) metabolites, respectively. The structure of M3, a less commonly seen carbamoyl glucuronide metabolite, was established using on-line H/D (hydrogen/deuterium) exchange experiments conducted by LC/MS. H/D exchange experiments were also used to distinguish the S-oxidation structures of M1 and M2 from hydroxylation. Herein, the application of deuterium oxide as the LC/MS mobile phase for structural elucidation of drug metabolites in biological matrices is demonstrated.

A rotaxane-like complex with controlled-release characteristics.

A rotaxane-like complex, based on a dumbbell-shaped component containing an NH(2)(+) recognition site for a [25]crown-8 ring component and a slippage stopper in the form of a p-(tert-butyl)phenyl group, has been synthesized by a "threading-followed-by-stoppering" approach. The half-life for dissociation of this complex, which is very sensitive to its environment, can be varied from minutes to months by changing the temperature and the polarity of the solvent.

Phosphorylated morpholine acetal human neurokinin-1 receptor antagonists as water-soluble prodrugs.

The regioselective dibenzylphosphorylation of 2 followed by catalytic reduction in the presence of N-methyl-D-glucamine afforded 2-(S)-(1-(R)-(3, 5-bis(trifluoromethyl)phenyl)ethoxy)-3-(S)-(4-fluoro)phenyl-4-(5-(2- phosphoryl-3-oxo-4H,-1,2,4-triazolo)methylmorpholine, bis(N-methyl-D-glucamine) salt, 11. Incubation of 11 in rat, dog, and human plasma and in human hepatic subcellular fractions in vitro indicated that conversion to 2 would be expected to occur in vivo most readily in humans during hepatic circulation. Conversion of 11 to 2 occurred rapidly in vivo in the rat and dog with the levels of 11 being undetectable within 5 min after 1 and 8 mg/kg doses iv in the rat and within 15 min after 0.5, 2, and 32 mg/kg doses iv in the dog. Compound 11 has a 10-fold lower affinity for the human NK-1 receptor as compared to 2, but it is functionally equivalent to 2 in preclinical models of NK-1-mediated inflammation in the guinea pig and cisplatin-induced emesis in the ferret, indicating that 11 acts as a prodrug of 2. Based in part on these data, 11 was identified as a novel, water-soluble prodrug of the clinical candidate 2 suitable for intravenous administration in humans.

Mycobacterium wolinskyi sp. nov. and Mycobacterium goodii sp. nov., two new rapidly growing species related to Mycobacterium smegmatis and associated with human wound infections: a cooperative study from the International Working Group on Mycobacterial Taxonomy.

Previous investigations demonstrated three taxonomic groups among 22 clinical isolates of Mycobacterium smegmatis. These studies were expanded to 71 clinical isolates, of which 35 (49%) (group 1) were identical to five ATCC reference strains including the type strain ATCC 19420T. Twenty-eight isolates (39%) were group 2, and eight isolates (11%) were group 3. Isolates of groups 2 and 3 were most often associated with post-traumatic or post-surgical wound infections including osteomyelitis, were susceptible to sulfamethoxazole, amikacin, imipenem and the tetracyclines, variably resistant to clarithromycin, and susceptible (group 1), intermediately resistant (group 2) or resistant (group 3) to tobramycin. The three groups were similar by routine biochemical and growth characteristics, but had different mycolic acid dimethoxy-4-coumarinylmethyl ester elution patterns by HPLC and different PCR-restriction enzyme patterns of a 439 bp fragment of the hsp-65 gene. Group 3 isolates differed from group 1 by 18 bp by 16S rRNA sequencing and exhibited < 25% homology by DNA-DNA hybridization, being most closely related to Mycobacterium mageritense. The 16S rRNA of group 1 and group 2 isolates differed by only 3 bp, but by DNA-DNA hybridization they exhibited only 40% homology. The following names are proposed: Mycobacterium goodii sp. nov. for group 2 isolates (type strain ATCC 700504T = MO69T), Mycobacterium wolinskyi sp. nov. for group 3 isolates (type strain ATCC 700010T = MO739T) and Mycobacterium smegmatis sensu stricto for group 1 isolates.

Substance P receptor antagonist I: conversion of phosphoramidate prodrug after i.v. administration to rats and dogs.

A water-soluble phosphoramidate prodrug (L-758,298, compound I) of the potent and selective human Substance P receptor antagonist L-754, 030 (compound II) is under development as an i.v. drug for treatment of emesis, migraine, and chronic pain. Compound I undergoes hydrolysis readily to II under acidic conditions. In the studies reported herein, we investigated the stability of I in blood and hepatic subcellular fractions from rats, dogs, and humans as well as the conversion of I to II in rats and dogs after i.v. dosing. Compound I was converted to II rapidly in rat blood but was stable in dog and human blood. However, the conversion was rapid in liver microsomes prepared from dogs and humans. As expected from the results of in vitro studies, the in vivo conversion of I to II was rapid after i.v. dosing of I to rats and dogs. The relative extent of exposure of II after i.v. dosing of I was estimated by comparing the dose-adjusted area under the plasma concentration versus time curve values of II after i.v. dosing of I with those after i.v. dosing of II. In rats, the extent of exposure was estimated to be approximately 90 and approximately 100% at 1 and 8 mg/kg, respectively; in dogs, that was approximately 59% at 0.5 mg/kg. A nonproportional increase in the area under the concentration versus time curve value of II with dose was observed after i.v. administration of I in dogs from 0.5 to 32 mg/kg, suggesting that the elimination of II might have been saturated at higher doses.

Mycophenolic acid increases apoptosis, lysosomes and lipid droplets in human lymphoid and monocytic cell lines.

BACKGROUND: Mycophenolic acid (MPA), a selective inhibitor of inosine monophosphate dehydrogenase, is the active agent of the immunosuppressive drug, mycophenolate mofetil (MMF). Previous studies have shown that MPA inhibits DNA synthesis in T and B lymphocytes by blocking de novo guanosine synthesis, and that MPA induces monocyte differentiation. MMF is being used for prevention of organ graft rejection and has also shown efficacy in rheumatoid arthritis trials. This study was designed to determine if apoptosis also plays a role in the immunosuppressive and anti-inflammatory effects of MMF. METHODS: Cultured human T lymphocytic (MOLT-4) and monocytic (THP-1 and U937) cell lines were treated with MPA. Apoptosis, cell viability, DNA content, lipid content, cell volume, and lysosomes were measured by a variety of microscopic, flow cytometric, and biochemical techniques. RESULTS: MPA inhibits proliferation, arrests cell cycle in S phase, and increases apoptosis in all three cell lines. Exogenous guanosine added within 24 hr of MPA treatment, but not later, partially reversed MPA-induced apoptosis in MOLT-4 cells. MPA increased lipid droplets in all three cell lines and increased both cell volumes and numbers of lysosomes in the monocytic cell lines. In both monocytic cell lines, MPA also reduced the number of nuclei containing nucleoli and greatly increased neutral lipids, primarily triacylglycerols, suggesting that these cells were differentiating. CONCLUSIONS: Increased apoptosis and terminal differentiation of both lymphocytes and monocytes may promote the antiproliferative, immunosuppressive, and anti-inflammatory effects of MMF seen clinically in transplantation and rheumatoid arthritis.

L-770,644: a potent and selective human beta3 adrenergic receptor agonist with improved oral bioavailability.

L-770,644 (9c) is a potent and selective agonist of the human beta3 adrenergic receptor (EC50 = 13 nM). It shows good oral bioavailability in both dogs and rats (%F = 27), and is a full agonist for glycerolemia in the rhesus monkey (ED50 = 0.21 mg/kg). Based on its desirable in vitro and in vivo properties, L-770,644 was chosen for further preclinical evaluation.

Studies on cytochrome P-450-mediated bioactivation of diclofenac in rats and in human hepatocytes: identification of glutathione conjugated metabolites.

The nonsteroidal anti-inflammatory drug diclofenac causes a rare but potentially fatal hepatotoxicity that may be associated with the formation of reactive metabolites. In this study, three glutathione (GSH) adducts, namely 5-hydroxy-4-(glutathion-S-yl)diclofenac (M1), 4'-hydroxy-3'-(glutathion-S-yl)diclofenac (M2), and 5-hydroxy-6-(glutathion-S-yl)diclofenac (M3), were identified by liquid chromatography-tandem mass spectrometry analysis of bile from Sprague-Dawley rats injected i.p. with a single dose of diclofenac (200 mg/kg). These adducts presumably were formed via hepatic cytochrome P-450 (CYP)-catalyzed oxidation of diclofenac to reactive benzoquinone imines that were trapped by GSH conjugation. In support of this hypothesis, M1, M2, and M3 were generated from diclofenac in incubations with rat liver microsomes in the presence of NADPH and GSH. Increases in adduct formation were observed when incubations were performed with liver microsomes from phenobarbital- or dexamethasone-treated rats. Adduct formation was inhibited by polyclonal antibodies against CYP2B, CYP2C, and CYP3A (40-50% inhibition at 5 mg of IgG/nmol of CYP) but not by an antibody against CYP1A. Maximal inhibition was obtained when the three inhibitory antibodies were used in a cocktail fashion (70-80% inhibition at 2.5 mg of each IgG/nmol of CYP). These data suggest that diclofenac undergoes biotransformation to reactive metabolites in rats and that CYP isoforms of the 2B, 2C, and 3A subfamilies are involved in this bioactivation process. With respect to CYP2C isoforms, rat hepatic CYP2C7 and CYP2C11 were implicated as mediators of the bioactivation based on immunoinhibition studies using antibodies specific to CYP2C7 and CYP2C11. Screening for GSH adducts also was carried out in human hepatocyte cultures containing diclofenac, and M1, M2, and M3 again were detected. It is possible, therefore, that reactive benzoquinone imines may be formed in vivo in humans and contribute to diclofenac-mediated hepatic injury.

Roles of human hepatic cytochrome P450s 2C9 and 3A4 in the metabolic activation of diclofenac.

Recently, it was shown that diclofenac was metabolized in rats to reactive benzoquinone imines via cytochrome P450-catalyzed oxidation. These metabolites also were detected in human hepatocyte cultures in the form of glutathione (GSH) adducts. This report describes the results of further studies aimed at characterizing the human hepatic P450-mediated bioactivation of diclofenac. The reactive metabolites formed in vitro were trapped by GSH and analyzed by LC/MS/MS. Thus, three GSH adducts, namely, 5-hydroxy-4-(glutathion-S-yl)diclofenac (M1), 4'-hydroxy-3'-(glutathion-S-yl)diclofenac (M2), and 5-hydroxy-6-(glutathion-S-yl)diclofenac (M3), were identified in incubations of diclofenac with human liver microsomes in the presence of NADPH and GSH. The formation of the adducts was taken to reflect the intermediacy of the corresponding putative benzoquinone imines. While M2 was the dominant metabolite over a substrate concentration range of 10-50 microM, M1 and M3 became equally important products at >/=100 microM diclofenac. The formation of M2 was inhibited by sulfaphenazole or an anti-P450 2C9 antibody (5-10% of control values). The formation of M1 and M3 was inhibited by troleandomycin, ketoconazole, or an anti-P450 3A4 antibody (30-50% of control values). In studies in which recombinant P450 isoforms were used, M2 was generated only by P450 2C9-catalyzed reaction, while M1 and M3 were produced by P450 3A4-catalyzed reaction. Good correlations were established between the extent of formation of M2 and P450 2C9 activities (r = 0.93, n = 10) and between the extent of formation of M1 and M3 and P450 3A4 activities (r = 0.98, n = 10) in human liver microsomal incubations. Taken together, the data suggest that the biotransformation of diclofenac to M2 is P450 2C9-dependent, whereas metabolism of the drug to M1 and M3 involves mainly P450 3A4. Although P450s 2C9 and 3A4 both catalyze the bioactivation of diclofenac, P450 2C9 is capable of producing the benzoquinone imine intermediate at lower drug concentrations which may be more clinically relevant.

Disposition of L-732,531, a potent immunosuppressant, in rats and baboons.

L-732,531 is a semi-synthetic analog of the macrolide tacrolimus (Prograf(R)). Like tacrolimus, L-732,531 is a potent immunosuppressant. In this study, its absorption, distribution, metabolism, and excretion were studied in rats and baboons. In rats, its blood and plasma levels were similar, whereas in baboons, its blood levels were, on average, twice as high as those in plasma. This was consistent with the in vitro blood-to-plasma ratio of L-732, 531, which in these two species, as well as in humans, was much lower than that of tacrolimus and showed a minimal concentration dependence. After iv administration to rats, the blood and plasma clearance of L-732,531 decreased from approximately 60 ml/min/kg at 0.2 mg/kg to 30 ml/min/kg when dosed at 1 and 3 mg/kg. After oral administration, plasma area under the concentration vs. time curve (AUC) and maximal plasma concentration (Cmax) increased more than proportionally to the dose. At 1, 5, and 15 mg/kg, plasma AUC was 29, 466, and 2832 ng.hr/ml, respectively, and Cmax was 10, 129, and 304 ng/ml, respectively. Bioavailability, although compromised by nonlinear kinetics, was estimated to be between 8% and 18%. In baboons, the clearance of L-732,531 was lower than that in rats, especially when calculated from blood concentrations (12 ml/min/kg at 0.2 mg/kg and 8 ml/min/kg at 1 mg/kg). After oral dosing, baboon plasma AUC and Cmax were much lower than those in rats, but as in rats, they increased more than proportionally with increasing doses. The bioavailability of L-732,531 in baboons was estimated at 3%, 9%, and 24% when animals were dosed at 5, 15, and 26 mg/kg po, respectively. After oral administration of [3H]L-732,531 at 5 mg/kg, approximately 32% of the radioactivity was recovered in bile and urine of rats, compared with 9% in baboons. High-performance liquid chromatography profiles of rat and baboon plasma, bile, urine, and feces indicated that L-732,531 was metabolized extensively to a complex mixture of products. Some intact parent drug was observed in feces of orally dosed animals, indicating incomplete absorption. In vitro, L-732,531 was metabolized more extensively by baboon liver microsomes than rat or human microsomes. Its metabolism in human liver microsomes was shown to be catalyzed primarily by cytochrome P450 3A isozymes.

Species differences in N-glucuronidation.

Glucuronidation of amines has been shown to exhibit species differences in vitro and in vivo. Substrates for N-glucuronidation can be classified according to the chemical structures of the resulting glucuronides into two groups: compounds that form non-quaternary N-conjugates, and those that form the quaternary counterparts. For compounds of the former class-such as sulfonamides, arylamines, and alicyclic, cyclic, and heterocyclic amines-species differences appear to be less striking and are of a quantitative nature. No one common laboratory animal species used routinely in metabolism research (e.g. rat, mouse, dog, non-human primate, rabbit, and guinea pig) has been shown to be deficient in N-glucuronidation when all of the substrates studied and reported are taken into consideration. The ability of a species to form N-glucuronides is compound-dependent, although rabbit and guinea pig appear to exhibit the highest capacity for this bioconjugation among preclinical species. For tertiary amines, most notably the tricyclic antidepressant and antihistamine drugs, N-glucuronidation is commonly observed in non-human primates and man. There are examples, however, of quaternary glucuronidation occurring in lower animal species. In exploring species differences in amine conjugation in vivo, it is noted that the apparent absence of N-glucuronides in animal urine may not reflect the inability of that species to form such conjugates, since the N-glucuronides may be excreted in bile. Problems such as degradation or low recoveries commonly encountered in isolation and identification of in vivo metabolites further complicate the interpretation of data. Because of the wide range of pKa values exhibited by various classes of amines, caution also should be exercised for in vitro studies since incubation conditions for N-glucuronidation often are substrate- and species-dependent. Explanations for the species differences observed in N-glucuronidation appear to be emerging as rapid advances are made in the understanding of the glucuronosyltransferases at the molecular level. More information, however, remains to be gathered from the glucuronosyltransferase genes of animal species other than humans before a better understanding of species differences in N-glucuronidation can be achieved.

A selective human beta3 adrenergic receptor agonist increases metabolic rate in rhesus monkeys.

Activation of beta3 adrenergic receptors on the surface of adipocytes leads to increases in intracellular cAMP and stimulation of lipolysis. In brown adipose tissue, this serves to up-regulate and activate the mitochondrial uncoupling protein 1, which mediates a proton conductance pathway that uncouples oxidative phosphorylation, leading to a net increase in energy expenditure. While chronic treatment with beta3 agonists in nonprimate species leads to uncoupling protein 1 up-regulation and weight loss, the relevance of this mechanism to energy metabolism in primates, which have much lower levels of brown adipose tissue, has been questioned. With the discovery of L-755,507, a potent and selective partial agonist for both human and rhesus beta3 receptors, we now demonstrate that acute exposure of rhesus monkeys to a beta3 agonist elicits lipolysis and metabolic rate elevation, and that chronic exposure increases uncoupling protein 1 expression in rhesus brown adipose tissue. These data suggest a role for beta3 agonists in the treatment of human obesity.