Supratherapeutic Psychotropic Drug Levels in the Emergency Department and Their Association with Delirium Duration: A Preliminary Study.

OBJECTIVES: Polypharmacy is associated with delirium, but the mechanisms for this connection are unclear. Our goal was to determine the frequency of supratherapeutic psychotropic drug levels (SPDLs) in older hospitalized patients and if it is associated with the duration of emergency department (ED) delirium. DESIGN: Secondary analysis of a prospective cohort study. SETTING: Tertiary care academic medical center. PARTICIPANTS: ED patients 65 years or older who were admitted to the hospital. MEASUREMENTS: Delirium was assessed in the ED and during the first 7 days of hospitalization using the modified Brief Confusion Assessment Method. Drug concentrations were determined in serum samples collected at enrollment via a novel platform based on liquid chromatography-tandem mass spectrometry capable of identifying and quantitating 78 clinically approved medications including opioids, benzodiazepines, antidepressants, antipsychotics, and amphetamines. Patients with serum psychotropic drug concentrations above established reference ranges were considered supratherapeutic and have a SPDL. We performed proportional odds logistic regression to determine if SPDLs were associated with ED delirium duration adjusted for confounders. Medical record review was performed to determine if the doses of medications associated with SPDLs were adjusted at hospital discharge. RESULTS: A total of 158 patients were enrolled; of these, 66 were delirious in the ED. SPDLs were present in 11 (17%) of the delirious and 4 (4%) of the non-delirious ED patients. SPDLs were significantly associated with longer ED delirium duration (adjusted proportional odds ratio = 6.0; 95% confidence interval = 2.1-17.3) after adjusting for confounders. Of the 15 medications associated with SPDLs, 9 (60%) were prescribed at the same or higher doses at the time of hospital discharge. CONCLUSION: SPDLs significantly increased the odds of prolonged ED delirium episodes. Approximately half of the medications associated with SPDLs were continued after hospital discharge at the same or higher doses. J Am Geriatr Soc 67:2387-2392, 2019.

Medication Exposure in Highly Adherent Psychiatry Patients.

Medication exposure is dependent upon many factors, the single most important being if the patient took the prescribed medication as indicated. To assess medication exposure for psychotropic and other medication classes, we enrolled 115 highly adherent psychiatry patients prescribed five or more medications. In these patients, we measured 21 psychotropic and 38 nonpsychotropic medications comprising a 59 medication multiplex assay panel. Strict enrollment criteria and reconciliation of the electronic health record medication list prior to study initiation produced a patient cohort that was adherent with 91% of their prescribed medications as determined by comparing medications detected empirically in blood to the electronic health record medication list. In addition, 13% of detected medications were not in the electronic health record medication list. We found that only 53% of detected medications were within the literature-derived reference range with 41% below and 6% above the reference range specific to each medication. When psychotropic medications were analyzed near trough-level, only sertraline was found to be within the literature-derived reference range for all patients tested. Concentrations of the remaining medications indicated extensive exposure below the reference range. This is the first study to empirically and comprehensively assess medication exposure obtained in comorbid polypharmacy patients, minimizing the important behavioral factor of adherence in the study of medication exposure. These data indicate that low medication exposure is extensive and must be considered when therapeutic issues arise, including the lack of response to medication therapy.

Assessment of Patient Medication Adherence, Medical Record Accuracy, and Medication Blood Concentrations for Prescription and Over-the-Counter Medications.

Importance: Inaccurate medication records and poor medication adherence result in incomplete knowledge of therapy for patients. Objective: To study accuracy of medical records and patient adherence by measuring blood concentrations of medications. Design, Setting, and Participants: This cross-sectional study validated a serum-based liquid chromatography-tandem mass spectrometry assay to simultaneously quantify 263 medications used for acute and chronic conditions. The assay panel was applied to 3 clinical patient cohorts: residual serum from 1000 randomly selected samples sent for routine clinical chemistry testing between April 8 and October 6, 2015 (residuals cohort), 50 prospectively enrolled patients in a gastroenterology clinic between March 1 and March 15, 2016, who were prescribed more than 5 medications (gastroenterology care cohort), and a convenience cohort of 296 patients with hypertension who sought care in an emergency department (ED care cohort) between July 1, 2012, and April 25, 2013. Integrated data analysis of the cohorts was performed from August 22 to November 29, 2017. Main Outcomes and Measures: Medication serum concentrations, electronic health record medication lists, and predicted drug interactions. Results: Of the 1346 total samples, 1000 came from the residuals cohort (640 women and 360 men; median age, 60 years [interquartile range (IQR), 44-71 years]), 50 from the gastroenterology care cohort (30 women and 20 men; median age, 66 years [IQR, 62-70 years]), and 296 from the ED care cohort (160 women and 136 men; median age, 59 years [IQR, 52-66 years]). Median medication adherence, defined as the subset of detected medications from the prescription record, was 83% (IQR, 50%-100%) in the residuals cohort, 100% (IQR, 84%-100%) in the gastroenterology care cohort, and 78% (IQR, 57%-100%) in the ED care cohort. Patients adherent to 1 medication were more often adherent to other medications. Among patients prescribed 3 medications or more, there were no significant associations between medication adherence and sex or number of prescribed medications, and there was a modest association between adherence and age. By comparing detected vs prescribed medications, we detected a median of 0 (IQR, 0-2) medications per patient that were not listed in the electronic health record in the residuals cohort, 1 (IQR, 0-2) medication per patient that was not listed in the electronic health record in the gastroenterology care cohort, and 1 (IQR, 0-2) medication per patient that was not listed in the electronic health record in the ED care cohort. A total of 435 patients (43.5%) in the residuals cohort had no discrepancy between the electronic health record and detected medication lists, 22 patients (44.0%) in the gastroenterology care cohort had no discrepancy between the electronic health record and detected medication lists, and 41 patients (13.9%) in the ED care cohort had no discrepancy between the electronic health record and detected medication lists. Half of adverse drug reaction alerts occurred among medications detected without prescription. Conclusions and Relevance: Comprehensive medication monitoring offers promise to improve adherence, the accuracy of medical records, and the safety for patients with polypharmacy.

Medication adherence, medical record accuracy, and medication exposure in real-world patients using comprehensive medication monitoring.

BACKGROUND: Poor adherence to medication regimens and medical record inconsistencies result in incomplete knowledge of medication therapy in polypharmacy patients. By quantitatively identifying medications in the blood of patients and reconciling detected medications with the medical record, we have defined the severity of this knowledge gap and created a path toward optimizing medication therapy. METHODS AND FINDINGS: We validated a liquid chromatography-tandem mass spectrometry assay to detect and/or quantify 38 medications across a broad range of chronic diseases to obtain a comprehensive survey of patient adherence, medical record accuracy, and exposure variability in two patient populations. In a retrospectively tested 821-patient cohort representing U.S. adults, we found that 46% of medications assessed were detected in patients as prescribed in the medical record. Of the remaining medications, 23% were detected, but not listed in the medical record while 30% were prescribed to patients, but not detected in blood. To determine how often each detected medication fell within literature-derived reference ranges when taken as prescribed, we prospectively enrolled a cohort of 151 treatment-regimen adherent patients. In this cohort, we found that 53% of medications that were taken as prescribed, as determined using patient self-reporting, were not within the blood reference range. Of the medications not in range, 83% were below and 17% above the lower and upper range limits, respectively. Only 32% of out-of-range medications could be attributed to short oral half-lives, leaving extensive exposure variability to result from patient behavior, undefined drug interactions, genetics, and other characteristics that can affect medication exposure. CONCLUSIONS: This is the first study to assess compliance, medical record accuracy, and exposure as determinants of real-world treatment and response. Variation in medication detection and exposure is greater than previously demonstrated, illustrating the scope of current therapy issues and opening avenues that warrant further investigation to optimize medication therapy.

Managing Psychotropic Medications in Complex, Real-World Patients Using Comprehensive Therapeutic Drug Monitoring.

There are multiple treatment options for depression, anxiety, psychosis, and other psychiatric disorders, and psychiatry patients are often comorbid with complex, polypharmacy treatment regimens. Unlike cardiovascular disease and diabetes, there are no readily available biomarkers to gauge treatment success with psychotropic medications, often resulting in subjective determination of medication therapy effectiveness. The physiochemical properties of psychiatric medications in general lend themselves to quantitative measurement in blood, offering an avenue to optimize treatment for each patient. Herein, we describe a novel application that employs comprehensive therapeutic drug monitoring of both psychiatric and nonpsychiatric medications to holistically personalize therapy for complex psychiatry patients.

Co-prescription trends in a large cohort of subjects predict substantial drug-drug interactions.

Pharmaceutical prescribing and drug-drug interaction data underlie recommendations on drug combinations that should be avoided or closely monitored by prescribers. Because the number of patients taking multiple medications is increasing, a comprehensive view of prescribing patterns in patients is important to better assess real world pharmaceutical response and evaluate the potential for multi-drug interactions. We obtained self-reported prescription data from NHANES surveys between 1999 and 2010, and confirm the previously reported finding of increasing drug use in the elderly. We studied co-prescription drug trends by focusing on the 2009-2010 survey, which contains prescription data on 690 drugs used by 10,537 subjects. We found that medication profiles were unique for individuals aged 65 years or more, with ≥98 unique drug regimens encountered per 100 subjects taking 3 or more medications. When drugs were viewed by therapeutic class, it was found that the most commonly prescribed drugs were not the most commonly co-prescribed drugs for any of the 16 drug classes investigated. We cross-referenced these medication lists with drug interaction data from Drugs.com to evaluate the potential for drug interactions. The number of drug alerts rose proportionally with the number of co-prescribed medications, rising from 3.3 alerts for individuals prescribed 5 medications to 11.7 alerts for individuals prescribed 10 medications. We found 22% of elderly subjects taking both a substrate and inhibitor of a given cytochrome P450 enzyme, and 4% taking multiple inhibitors of the same enzyme simultaneously. By examining drug pairs prescribed in 0.1% of the population or more, we found low agreement between co-prescription rate and co-discussion in the literature. These data show that prescribing trends in treatment could drive a large extent of individual variability in drug response, and that current pairwise approaches to assessing drug-drug interactions may be inadequate for predicting real world outcomes.

Minor compensatory changes in SAGE Mdr1a (P-gp), Bcrp, and Mrp2 knockout rats do not detract from their utility in the study of transporter-mediated pharmacokinetics.

Mdr1a-, Bcrp-, and Mrp2-knockout rats are a more practical species for absorption, distribution, metabolism, and excretion (ADME) studies than murine models and previously demonstrated expected alterations in the pharmacokinetics of various probe substrates. At present, gene expression and pathology changes were systematically studied in the small intestine, liver, kidney, and brain tissue from male SAGE Mdr1a, Bcrp, and Mrp2 knockout rats versus wild-type Sprague-Dawley controls. Gene expression data supported the relevant knockout genotype. As expected, Mrp2 knockout rats were hyperbilirubinemic and exhibited upregulation of hepatic Mrp3. Overall, few alterations were observed within 112 ADME-relevant genes. The two potentially most consequential changes were upregulation of intestinal carboxylesterase in Mdr1a knockouts and catechol-O-methyltransferase in all tissues of Bcrp knockout rats. Previously reported upregulation of hepatic Mdr1b P-glycoprotein in proprietary Wistar Mdr1a knockout rats was not observed in the SAGE counterpart investigated herein. Relative liver and kidney weights were 22-53% higher in all three knockouts, with microscopic increases in hepatocyte size in Mdr1a and Mrp2 knockout rats and glomerular size in Bcrp and Mrp2 knockouts. Increased relative weight of clearing organs is quantitatively consistent with reported increases in the clearance of drugs that are not substrates of the knocked-out transporter. Overall, SAGE knockout rats demonstrated modest compensatory changes, which do not preclude their general application to study transporter-mediated pharmacokinetics. However, until future studies elucidate the magnitude of functional change, caution is warranted in rare instances of extensive metabolism by catechol-O-methyltransferase in Bcrp knockouts and intestinal carboxylesterase in Mdr1a knockout rats, specifically for molecules with free catechol groups and esters subject to gut-wall hydrolysis.

Genome-wide screen for modulation of hepatic apolipoprotein A-I (ApoA-I) secretion.

Control of plasma cholesterol levels is a major therapeutic strategy for management of coronary artery disease (CAD). Although reducing LDL cholesterol (LDL-c) levels decreases morbidity and mortality, this therapeutic intervention only translates into a 25-40% reduction in cardiovascular events. Epidemiological studies have shown that a high LDL-c level is not the only risk factor for CAD; low HDL cholesterol (HDL-c) is an independent risk factor for CAD. Apolipoprotein A-I (ApoA-I) is the major protein component of HDL-c that mediates reverse cholesterol transport from tissues to the liver for excretion. Therefore, increasing ApoA-I levels is an attractive strategy for HDL-c elevation. Using genome-wide siRNA screening, targets that regulate hepatocyte ApoA-I secretion were identified through transfection of 21,789 siRNAs into hepatocytes whereby cell supernatants were assayed for ApoA-I. Approximately 800 genes were identified and triaged using a convergence of information, including genetic associations with HDL-c levels, tissue-specific gene expression, druggability assessments, and pathway analysis. Fifty-nine genes were selected for reconfirmation; 40 genes were confirmed. Here we describe the siRNA screening strategy, assay implementation and validation, data triaging, and example genes of interest. The genes of interest include known and novel genes encoding secreted enzymes, proteases, G-protein-coupled receptors, metabolic enzymes, ion transporters, and proteins of unknown function. Repression of farnesyltransferase (FNTA) by siRNA and the enzyme inhibitor manumycin A caused elevation of ApoA-I secretion from hepatocytes and from transgenic mice expressing hApoA-I and cholesterol ester transfer protein transgenes. In total, this work underscores the power of functional genetic assessment to identify new therapeutic targets.

Kainic acid-induced F-344 rat model of mesial temporal lobe epilepsy: gene expression and canonical pathways.

Mesial temporal lobe epilepsy (MTLE) is a severe neurological condition of unknown pathogenesis for which several animal models have been developed. To obtain a better understanding of the underlying molecular mechanisms and identify potential biomarkers of lesion progression, we used a rat kainic acid (KA) treatment model of MTLE coupled with global gene expression analysis to examine temporal (four hours, days 3, 14, or 28) gene regulation relative to hippocampal histopathological changes. The authors recommend reviewing the companion histopathology paper (Sharma et al. 2008) to get a better understanding of the work presented here. Analysis of filtered gene expression data using Ingenuity Pathways Analysis (Ingenuity Systems, http://www.ingenuity.com) revealed that a number of genes pertaining to neuronal plasticity (RhoA, Rac1, Cdc42, BDNF, and Trk), neurodegeneration (Caspase3, Calpain 1, Bax, a Cytochrome c, and Smac/Diablo), and inflammation/immune-response pathways (TNF-alpha, CCL2, Cox2) were modulated in a temporal fashion after KA treatment. Expression changes for selected genes known to have a role in neuronal plasticity were subsequently validated by quantitative polymerase chain reaction (qPCR). Notably, canonical pathway analysis revealed that a number of genes within the axon guidance signaling canonical pathway were up-regulated from Days 3 to 28, which correlated with aberrant mossy fiber (MF) sprouting observed histologically beginning at Day 6. Importantly, analysis of the gene expression data also identified potential biomarkers for monitoring neurodegeneration (Cox2) and neuronal/synaptic plasticity (Kalrn).

Increased cardiovascular risk associated with reduced kidney function.

BACKGROUND: Individuals with chronic kidney disease (CKD) are at substantial risk for cardiovascular mortality, but the risk associated with specific glomerular filtration rates (GFRs) is unknown. The objective of this study was to investigate the relationship between level of kidney function and the risk of cardiovascular mortality in a diverse population. METHODS AND RESULTS: This was a nonconcurrent cohort study of 34,982 ambulatory patients. Kidney function was entered into the model as a time-dependent variable to minimize misclassification and allow for improved estimate of the effect of decreasing GFR on cardiovascular mortality. The adjusted hazard ratio for cardiovascular mortality was 1.00 (95% CI 0.93-1.06) with an estimated GFR (eGFR) of 45-59; 1.77 (95% CI 1.65-1.89) with an eGFR 30-44; 3.75 (95% CI 3.47-4.06) with an eGFR 15-29, and 3.83 (95% CI 3.40-4.33) with an eGFR <15. CONCLUSION: We demonstrate a graded risk of cardiovascular mortality with decreasing GFR, with a marked increase with an eGFR <45 ml/min/1.73 m(2). These data also suggest that the availability of eGFR to physicians has had little impact on reducing the cardiovascular risk facing individuals with CKD. Our findings further highlight the public health significance of CKD and the importance of its early identification and management to reduce cardiovascular mortality.

Combination bortezomib and rituximab treatment affects multiple survival and death pathways to promote apoptosis in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a distinct histologic subtype of B cell non-Hodgkins lymphoma (NHL) associated with an aggressive clinical course. Inhibition of the ubiquitin-proteasome pathway modulates survival and proliferation signals in MCL and has shown clinical benefit in this disease. This has provided rationale for exploring combination regimens with B-cell selective immunotherapies such as rituximab. In this study, we examined the effects of combined treatment with bortezomib and rituximab on patient-derived MCL cell lines (Jeko, Mino, SP53) and tumor samples from patients with MCL where we validate reversible proteasome inhibition concurrent with cell cycle arrest and additive induction of apoptosis. When MCL cells were exposed to single agent bortezomib or combination bortezomib/rituximab, caspase dependent and independent apoptosis was observed. Single agent bortezomib or rituximab treatment of Mino and Jeko cell lines and patient samples resulted in decreased levels of nuclear NFkappaB complex(es) capable of binding p65 consensus oligonucleotides, and this decrease was enhanced by the combination. Constitutive activation of the Akt pathway was also diminished with bortezomib alone or in combination with rituximab. On the basis of in vitro data demonstrating additive apoptosis and enhanced NFkappaB and phosphorylated Akt depletion in MCL with combination bortezomib plus rituximab, a phase II trial of bortezomib-rituximab in patients with relapsed/refractory MCL is underway.

Strategic applications of toxicogenomics in early drug discovery.

Productivity issues facing the pharmaceutical industry are numerous, and the current challenges come in the face of an aging population and a demand for new and better medications. These challenges call for improvement in the drug discovery and development process, which paradoxically comes on the heels of remarkable scientific advances and in an era of great opportunity in medical science. Despite these advances, the pharmaceutical industry has yet to translate breakthroughs in new technologies, including genomics, into new drug therapies for unmet medical needs. The strategic application of toxicogenomics to the earliest stages of a drug discovery program offers a valuable opportunity to identify potential safety hurdles earlier than is the norm today. We propose that using genomics predictively (in vitro to predict outcomes in vivo and short-term studies in vivo to predict safety issues in longer studies) can assist in reducing inefficiency in the current paradigm, which is still heavily weighted on traditional endpoints from lengthy in vivo studies. Implementation of these strategies will assist in solving the current pharmaceutical pipeline productivity dilemma of long cycle times and unacceptable attrition rates in the preclinical drug discovery process.

Microarray probe mapping and annotation in cross-species comparative toxicogenomics.

Genomics-based tools, such as microarrays, do appear to offer promise in evaluating the relevance of one species to another in terms of molecular and cellular response to a given treatment. However, to fulfill this promise the individual end points (i.e., the genes, proteins, or mRNAs) measured in one species must be mapped to corresponding end points in another species. Several approaches, along with their strengths and weaknesses, are described in this chapter. A sequential approach is described that first makes use of a "Genome To Genome Through Orthology" method, where probe sequences for a given species are mapped into full-length sequences for that species, associated with the locus for those sequences and then into a second species by consulting orthology resources. The second step supplements these results by mapping the probe sequences for the given species into the best matching transcript from any organism, which then are mapped into the appropriate native locus and finally into the second species via an orthology resource. The results of this method are given for an experiment comparing the transcriptional response of canine liver to phenobarbital with that of rat liver.

Chronic microcystin exposure induces hepatocyte proliferation with increased expression of mitotic and cyclin-associated genes in P53-deficient mice.

Homozygous p53 deficient knockout mice were used to assess the role of p53 in tumor promotion by the protein phosphatase inhibitor and hepatic tumor promoter microcystin-LR (MCLR). More than 50% of human cancers bear mutations in the p53 gene, and in particular, p53 tumor suppressor gene mutations have been shown to play a major role in hepatocarcinogenesis. Trp53 homozygous (inactivated p53) and age-matched wild-type control mice were assigned to vehicle or MCLR-treated groups. MCLR or saline was administered daily for up to 28 days. RNA from the 28-day study was hybridized onto Mouse Genome GeneChip arrays. Selected RNA from 28 days and earlier time points was also processed for quantitative polymerase chain reaction (PCR). Livers from the 28-day, Trp53-deficient, MCLR group displayed greater hyperplastic and dysplastic changes morphologically and increases in Ki-67 and phosphohistone H3 (mitotic marker) immunoreactivity. Gene-expression analysis revealed significant increases in expression of cell-cycle regulation and cellular proliferation genes in the MCLR-treated, p53-deficient mutant mice compared to controls. These data suggest that regulation of the cell cycle by p53 is important in preventing the proliferative response associated with chronic, sublethal microcystin exposure, and therefore, conclude that p53 plays an important role in MCLR-induced tumor promotion.

Chronic kidney disease prevalence and rate of diagnosis.

BACKGROUND: Chronic kidney disease is a major public health problem. However, no study to date has estimated the prevalence of chronic kidney disease based on the clinical guidelines established by the National Kidney Foundation and few studies have explored the rate of diagnoses by primary care providers. SUBJECTS AND METHODS: Cross-sectional study of ambulatory patients in Rochester, NY. The purpose of this study was to estimate the prevalence of chronic kidney disease and the rate of primary caregiver diagnosis in ambulatory patients with chronic kidney disease. RESULTS: Among the 24,492 outpatients that had at least 2 glomerular filtration rate estimates > or =3 months apart, 6895 had an estimated glomerular filtration rate <60 mL/min/1.73 m2, indicating a 28.2% period prevalence of chronic kidney disease. The rate of clinical diagnosis among those with chronic kidney disease was 26.5% (95% confidence interval, 17.9 to 35.1), suggesting that 74% of patients with chronic kidney disease are undiagnosed. CONCLUSIONS: We demonstrate that the prevalence of chronic kidney disease is substantially higher in health-seeking individuals than in the general population. Moreover, we demonstrate that laboratory reporting of estimated glomerular filtration rate using the Modification of Diet in Renal Disease equation alone does not result in an optimal rate of clinical diagnosis.

Hepatic gene expression changes in mice associated with prolonged sublethal microcystin exposure.

Microcystin-LR (MCLR) is an acute hepatotoxicant and suspected carcinogen. Previous chronic studies have individually described hepatic morphologic changes, or alterations in the cytoskeleton, cell signaling or redox pathways. The objective of this study was to characterize chronic effects of MCLR in wild-type mice utilizing gene array analysis, morphology, and plasma chemistries. MCLR was given daily for up to 28 days. RNA from the 28-day study was hybridized onto mouse genechip arrays. RNA from 4 hours, 24 hours, 4 days, 1 day, and 28 days for selected genes was processed for quantitative-PCR. Increases in plasma hepatic enzyme activities and decreases in total protein, albumin and glucose concentrations were identified in MCLR-treated groups at 14 and 28 days. Histologically, marked hepatokaryomegaly was identified in the 14-day MCLR group with the addition of giant cells at 28 days. Major gene transcript changes were identified in the actin organization, cell cycle, apoptotic, cellular redox, cell signaling, albumin metabolism, and glucose homeostasis pathways, and the organic anion transport polypeptide system. Using toxicogenomics, we have identified key molecular pathways involved in chronic sublethal MCLR exposure in wild-type mice, genes participating in those critical pathways and related them to cellular and morphologic alterations seen in this and other studies.

Underlying mechanisms of pharmacology and toxicity of a novel PPAR agonist revealed using rodent and canine hepatocytes.

Marked species-specific responses to agonists of the peroxisome proliferator-activated alpha receptor (PPAR alpha) have been observed in rats and dogs, two species typically used to assess the potential human risk of pharmaceuticals in development. In this study, we used primary cultured rat and dog hepatocytes to investigate the underlying mechanisms of a novel PPAR alpha and -gamma coagonist, LY465608, relative to fenofibrate, a prototypical PPAR alpha agonist. As expected, rat hepatocytes incubated with these two agonists demonstrated an increase in peroxisome number as evaluated by electron microscopy, whereas the peroxisome number remained unchanged in dog hepatocytes. Biochemical analysis showed that rat hepatocytes responded to PPAR agonists with an induction of both peroxisomal and mitochondrial beta-oxidation (PBox and MBox) activities. Dog hepatocytes treated with both PPAR agonists, however, did not show increased PBox activity but did demonstrate increased MBox activity. Analysis of peroxisomal beta-oxidation gene expression markers by quantitative real-time PCR confirmed that PPAR agonists induced the peroxisomal enzymes, acyl-coenzyme A (CoA) oxidase (Acox), enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase (Ehhadh), and 3-ketoacyl-CoA thiolase (Acaa1) at the transcriptional level in rat hepatocytes, but not dog hepatocytes. Expression of mRNA for the mitochondrial beta-oxidation gene hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase (Hadhb), however, increased in both rat and dog hepatocytes, consistent with biochemical measurements of peroxisomal and mitochondrial beta-oxidation. Repeat-dose nonclinical safety studies of LY465608 revealed abnormities in mitochondrial morphology and evidence of single-cell necrosis following 30 days of dosing exclusively in dogs, but not in rats. Microarray analysis indicated that dog hepatocytes, but not rat hepatocytes, treated with LY465608 had an expression profile consistent with abnormalities in the regulation of cell renewal and death, oxidative stress, and mitochondrial bioenergetics, which may explain the canine-specific toxicity observed in vivo with this compound. This increased sensitivity to mitochondrial toxicity of canine hepatocytes relative to rat hepatocytes identified using gene expression was confirmed using the fluorescent indicator tetramethylrhodamine ethyl ester (TMRE) and flow cytometry. At doses of 0.1 microM LY465608, canine hepatocytes showed a greater shift in fluorescence indicative of mitochondrial damage than observed with rat hepatocytes treated at 10 microM. In summary, using rat and dog primary hepatocytes, we replicated the pharmacologic and toxicologic effects of LY465608 observed in vivo during preclinical development and propose an underlying mechanism for these species-specific effects.

Farnesoid X receptor agonist reduces serum asymmetric dimethylarginine levels through hepatic dimethylarginine dimethylaminohydrolase-1 gene regulation.

The farnesoid X receptor (FXR, NR1H4) is a bile acid-responsive nuclear receptor that plays critical roles in the transcriptional regulation genes involved in cholesterol, bile acid, triglyceride, and carbohydrate metabolism. By microarray analysis of hepatic genes from female Zucker diabetic fatty (ZDF) rats treated with the FXR agonist GW4064, we have identified dimethylarginine dimethylaminohydrolase-1 (DDAH1) as an FXR target gene. DDAH1 is a key catabolic enzyme of asymmetric dimethylarginine (ADMA), a major endogenous nitric-oxide synthase inhibitor. Sequence analysis of the DDAH1 gene reveals the presence of an FXR response element (FXRE) located 90 kb downstream of the transcription initiation site and within the first intron. Functional analysis of the putative FXRE demonstrated GW4064 dose-dependent transcriptional activation from the element, and we have demonstrated that the FXRE sequence binds the FXR-RXR heterodimer. In vivo administration of GW4064 to female ZDF rats promoted a dose-dependent and >6-fold increase in hepatic DDAH1 gene expression. The level of serum ADMA was reduced concomitantly. These findings provide a mechanism by which FXR may increase endothelium-derived nitric oxide levels through modulation of serum ADMA levels via direct regulation of hepatic DDAH1 gene expression. Thus, beneficial clinical outcomes of FXR agonist therapy may include prevention of atherosclerosis and improvement of the metabolic syndrome.

Gene expression analysis in a canine model of X-linked Alport syndrome.

Chronic kidney disease (CKD) often culminates in renal failure as a consequence of progressive interstitial fibrosis and is an important cause of illness and death in dogs. Identification of disease biomarkers and gene expression changes will yield valuable information regarding the specific biological pathways involved in disease progression. Toward these goals, gene expression changes in the renal cortex of dogs with X-linked Alport syndrome (XLAS) were examined using microarray technology. Extensive changes in inflammatory, metabolic, immune, and extracellular matrix biology were revealed in affected dogs. Statistical analysis showed 133 genes that were robustly induced or repressed in affected animals relative to age-matched littermates. Altered expression of numerous major histocompatibility complex (MHC) molecules suggests that the immune system plays a significant role in XLAS. Increased expression of COL4A1 and TIMP-1 at the end stage of disease supports the suggestion that expression increases in association with progression of fibrosis and confirms an observation of increased COL4A1 protein expression. Clusterin may function as one of the primary defenses of the renal cortex against progressive injury in dogs with XLAS, as demonstrated here by increased CLU gene expression. Cellular mechanisms that function during excess oxidative stress might also act to deter renal damage, as evidenced by alterations in gene expression of SOD1, ACO1, FDXR, and GPX1. This investigation provides a better understanding of interstitial fibrosis pathogenesis, and potential biomarkers for early detection, factors that are essential to discovering more effective treatments thereby reducing clinical illness and death due to CKD.

The fall in creatine levels and creatine kinase isozyme changes in the failing heart are reversible: complex post-transcriptional regulation of the components of the CK system.

Decreases in total creatine kinase (CK) activity and creatine [Cr] combine to limit the capacity of the failing heart to rapidly re-synthesize ATP (energy reserve). If the loss in energy reserve could be reversed, cardiac contractile reserve may be improved. Here we test whether these changes are reversible during recovery from heart failure. Left ventricular (LV) contractile function was measured in chronically instrumented conscious dogs with heart failure (CHF) induced by cardiac pacing for 3-4 weeks, and after recovery from heart failure (Recovery) (unpaced) for 5-6 weeks. LV contractile function and contractile reserve were depressed in CHF but returned to control in Recovery. CK capacity fell by 55% in CHF due to decreases in [Cr] (-39%) and CK activity (-25%), but was fully restored in Recovery. CK-B isozyme activity, protein (Western) and mRNA levels (real time PCR), respectively, were higher by 2-, 5.4- and 11-fold in CHF and higher by 3-, 2- and 2-fold in Recovery. CK-MM activity was decreased (-30%) in CHF but returned to normal levels during Recovery; CK-M protein was 30% lower in both CHF and Recovery even though there were no changes in mRNA levels. A similar pattern was found for mitochondrial CK (sMtCK). Deceases in CK activity and [Cr] in CHF are reversible. Decreases in CK-MM and sMtCK activities, but not the increases in CK-BB and CK-MB, also reversed. Neither the changes in protein nor mRNA levels for CK-B and CK-M correlated to their activities, suggesting that CK is under complex post-transcriptional regulation.