A physiologically based pharmacokinetic modeling approach to predict disease-drug interactions: suppression of CYP3A by IL-6.

Elevated cytokine levels are known to downregulate expression and suppress activity of cytochrome P450 enzymes (CYPs). Cytokine-modulating therapeutic proteins (TPs) used in the treatment of inflammation or infection could reverse suppression, manifesting as TP-drug-drug interactions (TP-DDIs). A physiologically based pharmacokinetic model was used to quantitatively predict the impact of interleukin-6 (IL-6) on sensitive CYP3A4 substrates. Elevated simvastatin area under the plasma concentration-time curve (AUC) in virtual rheumatoid arthritis (RA) patients, following 100 pg/ml of IL-6, was comparable to observed clinical data (59 vs. 58%). In virtual bone marrow transplant (BMT) patients, 500 pg/ml of IL-6 resulted in an increase in cyclosporine AUC, also in good agreement with the observed data (45 vs. 39%). In a different group of BMT patients treated with cyclosporine, the magnitude of interaction with IL-6 was underpredicted by threefold. The complexity of TP-DDIs highlights underlying pathophysiological factors to be considered, but these simulations provide valuable first steps toward predicting TP-DDI risk.

Development and optimisation of a duplex real-time reverse transcription quantitative PCR assay targeting the VP7 and NS2 genes of African horse sickness virus.

Nucleotide sequences of 52 South African isolates of African horse sickness virus (AHSV) collected during 2004-2005 and including viruses of all nine AHSV serotypes, were used to design and develop a duplex real-time reverse transcription quantitative PCR (RT-PCR) assay targeting the VP7 (S8) and NS2 (S9) genes of AHSV. The assay was optimized for detection of AHSV in fresh and frozen blood of naturally infected horses. Assay performance was enhanced using random hexamers rather than gene-specific primers for RT, and with denaturation of double-stranded RNA in the presence of random hexamers. The assay was efficient with a linear range of at least five orders of magnitude. The analytical sensitivity of the assay was 132 copies of the target genes (4125 copies per ml of blood), and the assay was at least 10-fold more sensitive than virus isolation on BHK-21 cells. The assay was also highly specific because it did not detect related orbiviruses, such as bluetongue and equine encephalosis viruses.

Naturally occurring and melengestrol acetate-associated reproductive tract lesions in zoo canids.

As husbandry practices have improved, safe and effective contraception for captive wildlife management has become a necessity. Melengestrol acetate (MGA), a synthetic progestin, is highly effective and has been used in many zoo species. Long-term use of MGA has been associated with uterine lesions in zoo felids, but effects in zoo canids have not been evaluated. This retrospective study documented spontaneously occurring lesions and investigated the impact of MGA on the reproductive health of zoo canids. Reproductive tracts from adult females were submitted by US zoos to the Association of Zoos & Aquariums' Wildlife Contraception Center Health Surveillance Program. Reproductive tracts were sampled and processed for histopathologic examination following standard protocols. Microscopic evaluations were performed without prior knowledge of MGA treatment status. Prevalence of uterine lesions was evaluated and compared between MGA-treated animals (n = 20) and control (untreated) animals (n = 61). Common lesions within the study population as a whole included endometrial hyperplasia (predominantly cystic) (53%), hydrometra (33%), and adenomyosis (25%). Treatment with MGA was a risk factor for endometrial hyperplasia, hydrometra, fibrosis, and adenomyosis. Uterine mineralization occurred exclusively in MGA-treated animals. Results indicate that MGA contraception can lead to lesions that may permanently impair the fertility of females. Therefore, if long-term contraception of zoo canids is necessary, the use of alternate methods of reproductive control such as gonadotropin-releasing hormone (GnRH) analogs or GnRH vaccines that reduce gonadal hormone exposure should be pursued.

First day review after uncomplicated phacoemulsification: is it necessary?

PURPOSE: To determine whether first day follow-up is necessary after routine uncomplicated phacoemulsification cataract surgery. METHODS: Data collected prospectively at day 1 postoperative review. RESULTS: In 510 consecutive cases, serious complications occurred in 8 (1.6%) (wound leak [4], corneal abrasion [2], iris prolapse [1], hyphema [1]). Intraocular pressure (IOP) >30 mmHg was found in 26 (5.1%) and was strongly associated with a diagnosis of pre-existing glaucoma or ocular hypertension (odds ratio [OR] 7.7). Symptoms of headache or ocular discomfort occurred in 40 (7.8%), mostly in association with raised IOP, and were also associated with pre-existing glaucoma or ocular hypertension (OR 4.7). Central corneal edema was found in 61 (12.0%). In the absence of corneal edema, IOP was >30 mmHg in only two cases (0.39%). CONCLUSIONS: Few sight-threatening complications were detected on the morning after an uncomplicated procedure. First day follow-up may be safely omitted if adequate patient counseling is undertaken and there is provision of adequate access to eye services. Review prior to discharge on the day of surgery would provide an opportunity to detect these few surgical complications and for counseling. A diagnosis of glaucoma or ocular hypertension is a risk factor for significantly raised next day IOP and these patients are more likely to experience postoperative discomfort. They may benefit from prophylactic treatment.

Where is T5? A survey of anaesthetists.

The extent of a regional block for Caesarean section must be tested and documented before surgery commences. In recent years a block to 'touch' that includes T5 has increasingly been considered the best predictive test for a pain-free Caesarean section. Our survey examines the consistency with which different anaesthetists identified the location of the T5 dermatome. Seventy-three anaesthetists were asked to mark a point on an anatomical picture to indicate where they would test for T5. Overall there was good agreement on the location of the T5 dermatome, but one in seven anaesthetists were inaccurate by two or more dermatomes. There were no statistically significant differences between the subgroups of senior house officer, specialist registrar and consultant anaesthetists. The knowledge of relevant dermatome levels should be an integral part of obstetric anaesthetic training.

Genital tract smooth muscle tumors are common in zoo felids but are not associated with melengestrol acetate contraceptive treatment.

In a survey of gynecologic lesions in female zoo felids conducted to determine if the widely used progestin contraceptive melengestrol acetate (MGA) had adverse effects, numerous leiomyomas and leiomyosarcomas were detected. This current study aimed to characterize the morphologic features of these tumors, determine their prevalence, and assess if MGA was a risk factor for their genesis. Genital tracts from 219 zoo felids representing 23 species were evaluated, and leiomyomas were detected in 24% of the felids. Leiomyomas were often multiple and occurred in the myometrium, ovary, or adjacent broad ligament. The risk of developing leiomyomas increased with age, but MGA treatment or parity had no effect. Five other felids had leiomyosarcomas. Leiomyosarcomas were distinguished from poorly demarcated leiomyomas by the presence of local invasion, metastasis, and cellular atypia, but necrosis and mitotic rate were not distinguishing criteria. Four of five felids with leiomyosarcomas had been treated with MGA. These results indicate that leiomyomas are common spontaneous lesions in the genital tracts of zoo felids and their genesis is not linked to MGA exposure. Whether progression to malignancy is promoted by MGA warrants further investigation.

Development and modification of a recombinant cell bioassay to directly detect halogenated and polycyclic aromatic hydrocarbons in serum.

Polycyclic and halogenated aromatic hydrocarbons (PAHs/HAHs) are a diverse group of widespread and persistent environmental contaminants that can cause a variety of detrimental effects in vertebrates. As most available methods to detect these contaminants are expensive, labor and time intensive, and require large amounts of tissue for extraction and analysis, several rapid mechanistically based bioassay systems have been developed to detect these chemicals. Here we describe application and optimization of a recently developed recombinant mouse cell bioassay system that responds to both PAHs and HAHs with the rapid induction of firefly luciferase for the detection of these chemicals in whole serum samples. This chemically activated luciferase expression (CALUX) bioassay has been modified to allow rapid (4-h) and direct analysis of small volumes (25-50 microl) of whole serum in a 96-well microtiter plate format without the need for solvent extraction. This bioassay can detect as little as 10 parts per trillion of the most potent HAH, 2,3,7,8-TCDD, and is also sensitive to other HAHs and PAHs. The use of simple procedures corrects for interplate and intraplate variability and the Ah receptor dependence of the induction response is accounted for by use of the antagonist 4-amino-3-methoxyflavone.

Bioactivation and covalent binding of hydroxyfluperlapine in human neutrophils: implications for fluperlapine-induced agranulocytosis.

The use of fluperlapine and the structurally related clozapine has been associated with the induction of agranulocytosis in humans. Unlike clozapine, fluperlapine is relatively resistant to chemical and biochemical oxidations. In this study we demonstrated that 7-hydroxyfluperlapine, the major metabolite of fluperlapine in humans, is oxidized to a reactive intermediate by HOCl and by myeloperoxidase in the presence of H(2)O(2) and Cl(-). This reactive intermediate was identified as an iminoquinone species with a M + 1 ion at m/z 324 by mass spectrometry. The iminoquinone intermediate was trapped by N-acetyl-L-cysteine (NAC) as well as GSH. NMR spectra of the NAC adducts indicated that the NAC was bound to the 6 and 9 positions of the aromatic ring. This is the same orientation as the binding of nucleophiles to the reactive metabolite of clozapine. We were able to use an antibody against clozapine to demonstrate that 7-hydroxyfluperlapine, but not fluperlapine itself, covalently modifies human myeloperoxidase. Furthermore, we demonstrated that 7-hydroxyfluperlapine is metabolized by activated neutrophils to a reactive intermediate that covalently binds to neutrophils. In the presence of NAC or GSH, such covalent binding was inhibited and the NAC or GSH adducts were formed. Thus, the reactivity and even the orientation of the binding of the reactive metabolite of 7-hydroxyfluperlapine is very similar to that of clozapine. These results provide a mechanism for the formation of a reactive metabolite of fluperlapine similar to clozapine that may explain its ability to induce agranulocytosis.

A comparison of the covalent binding of clozapine and olanzapine to human neutrophils in vitro and in vivo.

Covalent binding of a reactive metabolite of clozapine to neutrophils or their precursors is thought to play a role in the development of clozapine-induced agranulocytosis. Immunoblotting studies with an anti-clozapine antiserum detected covalent binding of clozapine to human neutrophils in vitro when HOCl was used to generate clozapine reactive metabolite (major clozapine adducts of 31, 49, 58, 78, 86, 126, 160, and 204 kDa). In addition, incubating neutrophils with clozapine and H2O2 (major clozapine adducts of 49 and 58 kDa) or clozapine, H2O2, and human myeloperoxidase (major clozapine adducts of 31, 49, 58, and 126 kDa) also resulted in covalent binding of clozapine to the neutrophils. The covalent binding of clozapine to neutrophils was inhibited by extracellular glutathione when HOCl, but not H2O2 was used to generate reactive metabolite. We found that the antiserum against clozapine also recognized olanzapine, an antipsychotic drug that forms a similar reactive metabolite to clozapine but has not been associated with induction of agranulocytosis. Repeating the in vitro experiments with olanzapine revealed that the major olanzapine-modified polypeptides had molecular masses of 96, 130-170, and 218 kDa. Only relatively low levels of 31, 49, and 58 kDa adducts were observed. Clozapine-modified polypeptides also were detected in neutrophils from patients being treated with clozapine. A major 58-kDa clozapine-modified polypeptide was detected in all patients tested. In contrast, no drug-modified polypeptides were detected in neutrophils from patients taking olanzapine. The differences in covalent binding exhibited by the two compounds and, in particular, the lack of olanzapine binding to human neutrophils in vivo may help to explain the difference in toxicity of these two drugs.

Interindividual variability in P450-dependent generation of neoantigens in halothane hepatitis.

Halothane hepatitis occurs because susceptible patients mount immune responses to trifluoroacetylated protein antigens, formed following cytochrome P450-mediated bioactivation of halothane to trifluoroacetyl chloride. In the present study, an in vitro approach has been used to investigate the cytochrome P450 isozyme(s) which catalyze neoantigen formation and to explore the protective role of non-protein thiols (cysteine and reduced glutathione). Significant levels of trifluoroacetyl protein antigens were generated when human liver microsomes, and also microsomes from livers of rats pre-treated with isoniazid, phenobarbital or beta-naphtoflavone, were incubated with halothane plus a nicotinamide adenine dinucleotidephosphate (NADPH) generating system. Immunoblotting studies revealed that the major trifluoroacetyl antigens expressed in vitro exhibited molecular masses of 50-55 kDa and included 60 and 80 kDa neoantigens recognized by antibodies from patients with halothane hepatitis. Much lower concentrations of halothane were required to produce maximal antigen generation in isoniazid-induced rat microsomes, as compared with phenobarbital or isosafrole-induced microsomes (0.5 vs 12.5 microl/ml). In isoniazid-induced microsomes, antigen generation was inhibited > 90% by the nucleophiles cysteine and glutathione and by the CYP2E1-selective inhibitors diallylsulfide and p-nitrophenol, but was unaffected by inhibitors of other P450 isozymes (furafylline, sulfaphenazole or triacetyloleandomycin). Neoantigen formation in six human liver microsomal preparations was inhibited in the presence of diallylsulfide, but not by furafylline, sulfaphenazole or triacetyloleandomycin, and exhibited marked variability which correlated with CYP2E1 levels. These results suggest that the balance between metabolic bioactivation by CYP2E1 and detoxication of reactive metabolites by cellular nucleophiles could be an important metabolic risk factor in halothane hepatitis.

Prevalence of and associated risk factors for shedding Cryptosporidium parvum oocysts and Giardia cysts within feral pig populations in California.

Populations of feral pigs (Sus scrofa) may serve as an environmental reservoir of Cryptosporidium parvum oocysts and Giardia sp. cysts for source water. We conducted a cross-sectional study to determine the prevalence of and associated demographic and environmental risk factors for the shedding of C. parvum oocysts and Giardia sp. cysts. Feral pigs were either live-trapped or dispatched from 10 populations located along the coastal mountains of western California, and fecal samples were obtained for immunofluorescence detection of C. parvum oocysts and Giardia sp. cysts. We found that 12 (5.4%) and 17 (7.6%) of 221 feral pigs were shedding C. parvum oocysts and Giardia sp. cysts, respectively. The pig's sex and body condition and the presence of cattle were not associated with the probability of the shedding of C. parvum oocysts. However, younger pigs (< or = 8 months) and pigs from high-density populations (> 2.0 feral pigs/km2) were significantly more likely to shed oocysts compared to older pigs (> 8 months) and pigs from low-density populations (< or = 1.9 feral pigs/km2). In contrast, none of these demographic and environmental variables were associated with the probability of the shedding of Giardia sp. cysts among feral pigs. These results suggest that given the propensity for feral pigs to focus their activity in riparian areas, feral pigs may serve as a source of protozoal contamination for surface water.

Cytochrome P450 mediated bioactivation of methyleugenol to 1'-hydroxymethyleugenol in Fischer 344 rat and human liver microsomes.

Cytochrome P450 mediated metabolism of methyleugenol to the proximate carcinogen 1'-hydroxymethyleugenol has been investigated in vitro. Kinetic studies undertaken in liver microsomes from control male Fischer 344 rats revealed that this reaction is catalyzed by high affinity (Km of 74.9 +/- 9.0 microM, Vmax of 1.42 +/- 0.17 nmol/min/nmol P450) and low affinity (apparent Km several mM) enzymic components. Studies undertaken at low substrate concentration (20 microM) with microsomes from livers of rats treated with the enzyme inducers phenobarbital, dexamethasone, isosafrole and isoniazid indicated that a number of cytochrome P450 isozymes can catalyze the high affinity component. In control rat liver microsomes, 1'-hydroxylation of methyleugenol (assayed at 20 microM substrate) was inhibited significantly (P < 0.05) by diallylsulfide (40%), p-nitrophenol (55%), tolbutamide (30%) and alpha-naphthoflavone (25%) but not by troleandomycin, furafylline, quinine or cimetidine. These results suggested that the reaction is catalyzed by CYP 2E1 and by another as yet unidentified isozyme(s) (most probably CYP 2C6), but not by CYP 3A, CYP 1A2, CYP 2D1 or CYP 2C11. Administration of methyleugenol (0-300 mg/kg/day for 5 days) to rats in vivo caused dose-dependent auto-induction of 1'-hydroxylation of methyleugenol in vitro which could be attributed to induction of various cytochrome P450 isozymes, including CYP 2B and CYP 1A2. Consequently, high dose rodent carcinogenicity studies are likely to over-estimate the risk to human health posed by methyleugenol. The rate of 1'-hydroxylation of methyleugenol in vitro in 13 human liver samples varied markedly (by 37-fold), with the highest activities being similar to the activity evident in control rat liver microsomes. This suggests that the risk posed by dietary ingestion of methyleugenol could vary markedly in the human population.

Immunochemical detection of covalently modified protein adducts in livers of rats treated with methyleugenol.

Methyleugenol is an allylbenzene food flavoring which has been shown to form DNA and protein adducts, and to cause hepatotoxicity and carcinogenicity in rodents. In order to investigate the nature of the protein adducts, specific antisera were raised by immunizing rabbits with conjugates prepared by coupling 1'-acetoxymethyleugenol, or its acidic congener 3,4-dimethoxycinnamic acid, to rabbit serum albumin (RSA). These polyclonal antisera were shown by enzyme linked immunosorbent assay (ELISA) to contain antibodies which recognized the 3,4-dimethoxyphenyl ring portion of methyleugenol. Analysis of livers from rats given methyleugenol i.p. for 5 days, at doses between 10 and 300 mg/kg/day, revealed dose-dependent formation of novel protein adducts which were recognized by the antisera. The adducts were detected by ELISA and by immunoblotting and were concentrated in the microsomal fraction, and were shown in inhibition studies to be derived from methyleugenol. A 44 kDa adduct was the only protein adduct detected in livers of rats given low loses of methyleugenol (10 or 30 mg/kg/day) and was the major adduct detected in rats given high doses of the compound (100 and 300 mg/kg/day). This adduct was solubilized when microsomal fractions were extracted using 0.1 M sodium carbonate, implying that it is a peripheral membrane protein. A pattern of protein adducts which mirrored the in vivo situation was generated when rat hepatocytes were incubated with 1'-hydroxymethyleugenol in vitro, but could not be reproduced in experiments undertaken using liver microsomes or postmitochondrial supernatants. These findings imply that generation of protein adducts in livers of rats given methyleugenol in vivo proceeds via the 1'-hydroxy metabolite and requires crucial cofactors, and/or structural features, which are present in intact hepatocytes but not in broken cell preparations and which remain to be defined.

Pregnancy detection in bighorn sheep (Ovis canadensis) using a fecal-based enzyme immunoassay.

We developed and validated an enzyme immunoassay for immunoreactive pregnanediol-3-glucuronide (iPdG) in feces to monitor reproductive status in desert and Rocky Mountain bighorn sheep (Ovis canadensis). Fecal iPdG concentrations were strongly correlated (r = 0.71) with serum progesterone concentrations in paired fecal and blood samples collected from 34 free-ranging desert bighorn sheep. In bimonthly fecal samples collected from 12 captive ewes, fecal iPdG profiles were similar between desert and Rocky Mountain bighorn sheep and we selected a pregnancy detection cutoff value of iPdG > or = 1.8 ng/mg feces. Fecal iPdG concentrations always exceeded this cutoff value when samples were collected from about day 60 of pregnancy to a few days before parturition, but values < 1.8 ng/mg (false negatives) were common for samples collected during the first 60 days of gestation. Although we tested a small number of known pregnant and non-pregnant ewes, the accuracy of the assay was 100% when two samples, collected 2 wk apart, were evaluated for any given ewe. Based on these data, this direct enzyme immunoassay for fecal progesterone metabolites has promise as a diagnostic tool to monitor hormone excretion and pregnancy in a free-ranging ungulate species.

Microvascular permeability and endothelial cell morphology associated with low-flow ischemia/reperfusion injury in the equine jejunum.

Microvascular permeability of the jejunum of clinically normal equids and microvascular permeability associated with 60 minutes of ischemia (25% baseline blood flow) and subsequent reperfusion were investigated. Eight adult horses were randomly allotted to 2 equal groups: normal and ischemic/reperfusion injury. Lymphatic flow rates, mesenteric blood flow, and lymph and plasma protein concentrations were determined at 15-minute intervals throughout the study. Microvascular permeability was determined by estimates of the osmotic reflection coefficient, which was determined when the ratio of lymphatic protein to plasma protein concentration reached a constant minimal value as lymph flow rate increased (filtration-independent lymph flow rate), which occurred at venous pressure of 30 mm of Hg. Full-thickness jejunal biopsy specimens were obtained at the beginning and end of each experiment, and were prepared for light microscopy to estimate tissue volume (edema) and for transmission electron microscopy to evaluate capillary endothelial cell morphology. The osmotic reflection coefficient for normal equine jejunum was 0.19 +/- 0.06, and increased significantly (P < or = 0.0001) to 0.48 +/- 0.05 after the ischemia/reperfusion period. Microscopic evaluation revealed a significant increase (P < or = 0.0001) in submucosal and serosal volume and capillary endothelial cell damage in horses that underwent ischemia/reperfusion injury. Results indicate that ischemia/reperfusion of the equine jejunum caused a significant increase in microvascular permeability.

Performance of various tests used to screen antibiotic residues in milk samples from individual animals.

The 10-point Milk and Dairy Beef Quality Assurance Program was developed collaboratively by the National Milk Producers Federation and the American Veterinary Medical Association and is designed to promote and document the responsible use of antibiotics in the dairy industry. One area of emphasis in this program is testing of individual animals for antibiotic residues after a specified post-treatment withdrawal time. We examined the performance of various assay systems on milk samples from individual cows. These assays are used at present on bulk tank milk samples by regulatory agencies, processing plants, producers, and veterinarians to detect the presence of beta-lactam antibiotics. A high proportion of false-positive results was obtained for both the pretreatment milk samples from cows with clinical mastitis and the milk samples obtained 21 days after initial therapy (nonantibiotic and antibiotic) for the treatment of mastitis. A high proportion of false-positive outcomes was obtained from the milk of clinically normal cows that had not received any medication for at least 30 days prior to evaluation. The results indicate a serious problem in the use of some assays that were designed to evaluate residues bulk tank milk samples to analyze samples from individual cows. This error in assay specificity results in the unjustifiable discarding of milk that meets regulatory standards and may be misused to accuse the producer or veterinarian of not adhering to regulatory guidelines. Maintaining a safe, high-quality milk supply is a constant goal of the dairy industry, which must be provided the appropriate tools and techniques to meet this challenge.