Application of a probabilistic method for the determination of drug-induced QT prolongation in telemetered cynomolgus monkeys: effects of moxifloxacin.

INTRODUCTION: Moxifloxacin is the most widely used positive reference agent in clinical cardiac repolarization safety studies, but it has not been characterized in the cynomolgus monkey. This important experimental animal species exhibits pronounced heart rate variability, complicating the temporal evaluation of QT interval data. METHODS: Digitized epicardial ECGs and aortic blood pressures were collected for 20 h in telemetered cynomolgus monkeys (n=6) following the administration of either vehicle or moxifloxacin (10 or 50 mg/kg, p.o.). Moxifloxacin plasma concentrations were determined 4 h postdose. ECG intervals were analyzed by computerized algorithms. Individual probabilistic QT rate-corrections (QTc) were derived from the slopes of predose log-transformed QT-RR data where each QT value was the mean of >250 beats/RR increment. The resulting QTc was used to determine the repolarization effects of moxifloxacin, expressed as the placebo-adjusted change in QTc (DeltaQTc), and as the integrated response from 0 to 12 h (AUC(0-->12)) postdose. RESULTS: No DeltaQTc effect was produced by 10 mg/kg moxifloxacin. However, moxifloxacin (50 mg/kg; 5.86+/-0.5 microg/mL C(max)) significantly prolonged the RR interval by 50 to 112 ms from 3.5 to 7.5 h postdose and DeltaQTc by >or=7.2 ms from 1.83 to 9.17 h, with a maximal DeltaQTc effect of +26.4 ms. No notable effects on either systemic blood pressure or body temperature occurred with either dose. DISCUSSION: Probabilistic QT rate-corrections appear to have eliminated the confounding effects of heart rate, provided for a stable QTc baseline, and enabled the demonstration of an exposure-dependent QTc prolongation by moxifloxacin. The duration and magnitude of the QTc effect paralleled moxifloxacin pharmacokinetics, and C(max) values were similar to those achieved clinically in thorough QT/QTc studies. Thus, novel probabilistic QT rate-corrections may offer highly robust assessments of repolarization risk in both nonclinical and clinical investigations.

Novel probabilistic method for precisely correcting the QT interval for heart rate in telemetered dogs and cynomolgus monkeys.

INTRODUCTION: QT intervals are not regulated on a beat-to-beat cadence, but are strongly influenced by the preceding heart rate history (hysteresis). ECG sampling, when performed over sufficiently long periods, results in the detection of ranges of different QT values for each discrete RR interval. Given the potential impact of QT hysteresis in QT interval rate-correction procedures, we hypothesized that, physiologically, the QT interval exists as a probabilistic variable where the exact value corresponding to any RR interval is precisely estimated from the associated QT population. METHODS: Digital ECGs were collected for 18-21 h in telemetered dogs (n=7) and cynomolgus monkeys (n=7) employing epicardial ECG leads for accurate T(end) detection, and analyzed by computerized algorithms. Descriptive statistics were calculated for raw QT values in 10 ms RR increments. Individual rate-corrected QT (QTc) formulae were derived from the slopes of log-transformed QT-RR data where each QT point was the mean of >250 beats/RR increment. The aptness of this QTc model was assessed by residual analysis. RESULTS: Beat-to-beat ECG analysis demonstrated that for all discrete cycle lengths, the associated raw QT intervals were normally distributed populations, spanning approximately 30-40 and 45-100 ms in the dog and cynomolgus monkey, respectively. In both species, QTc was stable (< or =5 ms variation) over all physiological RR intervals. DISCUSSION: The probabilistic treatment of raw QT interval populations natively associated to any RR interval provides hysteresis-free raw QT estimates which can be accurately modeled, allowing the derivation of a precise QTc value. Previous unawareness of the probabilistic nature of the QT interval explains the historical failure of numerous QT rate-correction formulae to correctly solve this scientific issue. Importantly, QT distribution analysis has the potential to provide, for the first time, a universal and sensitive method for QT heart rate-correction, providing a robust method for nonclinical and clinical cardiac safety investigations of repolarization delay.

Lobucavir-induced proliferative changes in mice.

Nucleoside analogues are used in the treatment of viral infections, including those caused by human immunodeficiency virus, cytomegalovirus, and herpes virus. These drugs are beneficial in the treatment of human disease, but are associated with toxicities that often limit their intended therapeutic use, including anemia, neutropenia, peripheral neuropathy, and myopathy. Some of these compounds have been reported to be carcinogenic in rodents. To investigate the carcinogenic potential of lobucavir, a nucleoside analogue, three groups of 60 male and female mice were orally administered lobucavir at daily doses of 10, 50, and 250 mg/kg (males) or 30, 150, and 750 mg/kg (females) over a period of 104 weeks. Two identical groups of 60 male and female mice each served as controls. The morphology and the incidence of neoplasms is described and compared with the tumor spectrum of other nucleoside analogues. Light microscopically, lobucavir-induced neoplastic lesions consisted of upper digestive tract squamous cell neoplasia in males and females; cervical, vaginal, and cutaneous squamous cell neoplasia in females; and Hardarian gland adenomas and adenocarcinomas in male mice. These results suggest that long-term administration of lobucavir causes neoplasia in mice, the spectrum of which resembles that observed after long-term administration of zidovudine or ganciclovir.

Distinct roles of NF-kappaB p50 in the regulation of acetaminophen-induced inflammatory mediator production and hepatotoxicity.

Oxidative stress plays an important role in acetaminophen (APAP)-induced hepatotoxicity. In addition to inducing direct cellular damage, oxidants can activate transcription factors including NF-kappaB, which regulate the production of inflammatory mediators implicated in hepatotoxicity. Here, we investigated the role of APAP-induced oxidative stress and NF-kappaB in inflammatory mediator production. Treatment of mice with APAP (300 mg/kg, i.p.) resulted in centrilobular hepatic necrosis and increased serum aminotransferase levels. This was correlated with depletion of hepatic glutathione and CuZn superoxide dismutase (SOD). APAP administration also increased expression of the proinflammatory mediators, interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNFalpha), macrophage chemotactic protein-1 (MCP-1), and KC/gro, and the anti-inflammatory cytokine, interleukin-10 (IL-10). Pretreatment of mice with the antioxidant, N-acetylcysteine (NAC) prevented APAP-induced depletion of glutathione and CuZnSOD, as well as hepatotoxicity. NAC also abrogated APAP-induced increases in TNFalpha, KC/gro, and IL-10, but augmented expression of the anti-inflammatory cytokines interleukin-4 (IL-4) and transforming growth factor-beta (TGFbeta). No effects were observed on IL-1beta or MCP-1 expression. To determine if NF-kappaB plays a role in regulating mediator production, we used transgenic mice with a targeted disruption of the gene for NF-kappaB p50. As observed with NAC pretreatment, the loss of NF-kappaB p50 was associated with decreased ability of APAP to upregulate TNFalpha, KC/gro, and IL-10 expression and increased expression of IL-4 and TGFbeta. However, in contrast to NAC pretreatment, the loss of p50 had no effect on APAP-induced hepatotoxicity. These data demonstrate that APAP-induced cytokine expression in the liver is influenced by oxidative stress and that this is dependent, in part, on NF-kappaB. However, NF-kappaB p50-dependent responses do not appear to play a major role in the pathogenesis of toxicity in this model.

Probabilistic neural network multiple classifier system for predicting the genotoxicity of quinolone and quinoline derivatives.

Quinolone and quinoline are known to be liver carcinogens in rodents, and a number of their derivatives have been shown to exhibit mutagenicity in the Ames test, using Salmonella typhimurium strain TA 100 in the presence of S9. Both the carcinogenicity and the mutagenicity of quinolone and quinoline derivatives, as determined by SAS, can be attributed to their genotoxicity potential. This potential, which is measured by genotoxicity tests, is a good indication of carcinogenicity and mutagenicity because compounds that are positive in these tests have the potential to be human carcinogens and/or mutagens. In this study, a collection of quinolone and quinoline derivatives' carcinogenicity is determined by qualitatively predicting their genotoxicity potential with predictive PNN (probabilistic neural network) classification models. In addition, a multiple classifier system is also developed to improve the predictability of genotoxicity. Superior results are seen with the multiple classifier system over the individual PNN classification models. With the multiple classifier system, 89.4% of the quinolone derivatives were predicted correctly, and higher predictability is seen with the quinoline derivatives at 92.2% correct. The multiple classifier system not only is able to accurately predict the genotoxicity but also provides an insight about the main determinants of genotoxicity of the quinolone and quinoline derivatives. Thus, the PNN multiple classifier system generated in this study is a beneficial contributor toward predictive toxicology in the design of less carcinogenic bioactive compounds.

Development of the ICH guidelines for immunotoxicology evaluation of pharmaceuticals using a survey of industry practices.

An anonymous survey of pharmaceutical industry practices for immunotoxicology evaluation was conducted. This was in support of the development of the guideline on the preclinical evaluation of unintended modulation of the immune system for the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use. The survey was conducted in two phases in 2003 and 2004. A total of 64 responses were received of which 45 were included in the formal evaluation. The remaining compounds were excluded because they were cytotoxic anti-neoplastic drugs (N = 7), or due to insufficient information (N = 12). The purpose of the survey was to gather data on the correlation between routine toxicology studies (RTS) and additional immunotoxicological studies (AIS). The results of the survey were evaluated by the Expert Working Group (EWG) and classified as to positive or negative findings in RTS and AIS. The results of the survey showed that for 27 of 45 compounds (60%), the RTS and AIS endpoints were in agreement. In 12 of 45 cases (27%), the RTS endpoints showed immune modulation not observed in the AIS assays. Finally for 6 of 45 drugs (13%) a response was seen with the AIS methods where no significant effect was observed in the RTS endpoints. Length of dosing and the number of tests evaluated were similar in all groups. The groups where RTS detected signs of immunosuppression were more likely to have been dosed at or above MTD. This data contributed to the consensus in the EWG that routine immune function testing as an initial screen for all new drugs is not required. Instead, a weight-of-evidence approach including RTS and other causes for concern is recommended to identify the need for additional immunotoxicity studies.

Predicting the genotoxicity of polycyclic aromatic compounds from molecular structure with different classifiers.

Classification models were developed to provide accurate prediction of genotoxicity of 277 polycyclic aromatic compounds (PACs) directly from their molecular structures. Numerical descriptors encoding the topological, geometric, electronic, and polar surface area properties of the compounds were calculated to represent the structural information. Each compound's genotoxicity was represented with IMAX (maximal SOS induction factor) values measured by the SOS Chromotest in the presence and absence of S9 rat liver homogenate. The compounds' class identity was determined by a cutoff IMAX value of 1.25-compounds with IMAX > 1.25 in either test were classified as genotoxic, and the ones with IMAX < or = 1.25 were nongenotoxic. Several binary classification models were generated to predict genotoxicity: k-nearest neighbor (k-NN), linear discriminant analysis, and probabilistic neural network. The study showed k-NN to provide the highest predictive ability among the three classifiers with a training set classification rate of 93.5%. A consensus model was also developed that incorporated the three classifiers and correctly predicted 81.2% of the 277 compounds. It also provided a higher prediction rate on the genotoxic class than any other single model.

Role of CCR2 in macrophage migration into the liver during acetaminophen-induced hepatotoxicity in the mouse.

The biological effects of monocyte chemoattractant protein (MCP) 1 are mediated by binding to C-C chemokine receptor (CCR) 2. In the present studies, we used CCR2 knockout (CCR2-/-) mice to examine the role of MCP-1 in acetaminophen-induced macrophage accumulation in the liver, expression of inflammatory cytokines, and hepatotoxicity. We found that hepatic expression of CCR2 and MCP-1 was increased 10-fold and 20-fold, respectively, 12 to 72 hours after administration of acetaminophen to wild-type mice. Expression of these proteins was localized in centrilobular regions of the liver. Whereas MCP-1 was expressed by both hepatocytes and macrophages, CCR2 was identified in inflammatory macrophages. F4/80 is a marker of mature macrophages expressed in large quantities by Kupffer cells. In wild-type mice, a 75% decrease in F4/80-positive macrophages was observed 24 to 48 hours after administration of acetaminophen. In contrast, expression of macrosialin (CD68), a marker of activated macrophages, increased 2-fold 24 to 72 hours after administration of acetaminophen and was associated with inflammatory cells. Although there was a decrease in the overall severity of inflammation and in the number of macrosialin-positive macrophages 72 hours after administration of acetaminophen in CCR2-/- mice, the number of F4/80-positive cells did not change. Loss of CCR2 was also found to alter acetaminophen-induced expression of tumor necrosis factor alpha, monocyte chemoattractant protein 3, and KC/gro. However, the overall outcome of acetaminophen-induced hepatic injury was not affected. In conclusion, these data indicate that MCP-1 and CCR2 contribute to the recruitment of a subset of activated macrophages into the liver during acetaminophen-induced hepatotoxicity that may be important in resolution of tissue injury.

Predicting the genotoxicity of secondary and aromatic amines using data subsetting to generate a model ensemble.

Binary quantitative structure-activity relationship (QSAR) models are developed to classify a data set of 334 aromatic and secondary amine compounds as genotoxic or nongenotoxic based on information calculated solely from chemical structure. Genotoxic endpoints for each compound were determined using the SOS Chromotest in both the presence and absence of an S9 rat liver homogenate. Compounds were considered genotoxic if assay results indicated a positive genotoxicity hit for either the S9 inactivated or S9 activated assay. Each compound in the data set was encoded through the calculation of numerical descriptors that describe various aspects of chemical structure (e.g. topological, geometric, electronic, polar surface area). Furthermore, five additional descriptors that focused on the secondary and aromatic nitrogen atoms in each molecule were calculated specifically for this study. Descriptor subsets were examined using a genetic algorithm search engine interfaced with a k-Nearest Neighbor fitness evaluator to find the most information-rich subsets, which ultimately served as the final predictive models. Models were chosen for their ability to minimize the total number of misclassifications, with special attention given to those models that possessed fewer occurrences of positive toxicity hits being misclassified as nontoxic (false negatives). In addition, a subsetting procedure was used to form an ensemble of models using different combinations of compounds in the training and prediction sets. This was done to ensure that consistent results could be obtained regardless of training set composition. The procedure also allowed for each compound to be externally validated three times by different training set data with the resultant predictions being used in a "majority rules" voting scheme to produce a consensus prediction for each member of the data set. The individual models produced an average training set classification rate of 71.6% and an average prediction set classification rate of 67.7%. However, the model ensemble was able to correctly classify the genotoxicity of 72.2% of all prediction set compounds.

Predicting the genotoxicity of thiophene derivatives from molecular structure.

We report several binary classification models that directly link the genetic toxicity of a series of 140 thiophene derivatives with information derived from the compounds' molecular structure. Genetic toxicity was measured using an SOS Chromotest. IMAX (maximal SOS induction factor) values were recorded for each of the 140 compounds both in the presence and in the absence of S9 rat liver homogenate. Compounds were classified as genotoxic if IMAX >or= 1.5 in either test or nongenotoxic if IMAX < 1.5 for both tests. The molecular structures were represented by numerical descriptors that encoded the topological, geometric, electronic, and polar surface area properties of the thiophene derivatives. The classification models used were linear discriminant analysis (LDA), k-nearest neighbor classification (k-NN), and the probabilistic neural network (PNN). These were used in conjunction with either a genetic algorithm or a generalized simulated annealing to find optimal subsets of descriptors for each classifier. The quality of the resulting models was determined by the number of misclassified compounds, with preference given to models that produced fewer false negative classifications. Model sizes ranged from seven descriptors for LDA to three descriptors for k-NN and PNN. Very good classification results were obtained with all three classifiers. Classification rates for the LDA, k-NN, and PNN models were 80, 85, and 85%, respectively, for the prediction set compounds. Additionally, a consensus model was generated that incorporated all three of the basic model types. This consensus model correctly predicted the genotoxicity of 95% of the prediction set compounds.

Role of tumor necrosis factor receptor 1 (p55) in hepatocyte proliferation during acetaminophen-induced toxicity in mice.

Hepatocyte proliferation represents an important part of tissue repair. In these studies, TNF receptor 1 (TNFR1) knockout mice were used to analyze the role of TNF-alpha in hepatocyte proliferation during acetaminophen-induced hepatotoxicity. Treatment of wild-type (WT) mice with acetaminophen (300 mg/kg) resulted in centrilobular hepatic necrosis. This was associated with proliferation of hepatocytes surrounding the damaged areas, which was evident at 24 h. The cell cycle regulatory proteins, cyclin D1 and cyclin A, were also up regulated in hepatocytes. In contrast, in TNFR1-/- mice, which exhibit exaggerated acetaminophen hepatotoxicity, hepatocyte proliferation, and expression of cyclin D1 and cyclin A, as well as the cyclin dependent kinases, Cdk4 and Cdk2, were reduced. The cyclin-dependent kinase inhibitor p21 was also induced in the liver following acetaminophen administration. This was greater in TNFR1-/- mice compared to WT mice. To investigate mechanisms mediating the reduced hepatic proliferative response of TNFR1-/- mice, we analyzed phosphatidyl inositol-3-kinase (PI-3K) signaling. In both WT and TNFR1-/- mice, acetaminophen caused a rapid increase in total PI-3K within 3 h. Acetaminophen also increased phosphorylated PI-3K, but this was delayed 6-12 h in TNFR1-/- mice. Expression of Akt, a downstream target of PI-3K, was increased in both WT and TNFR1-/- mice in response to acetaminophen. However, the increase was greater in WT mice. Acetaminophen-induced expression of phosphorylated STAT3, a key regulator of cytokine-induced hepatocyte proliferation, was also delayed in TNFR1-/- mice relative to WT. These data suggest that TNF-alpha signaling through TNFR1 is important in regulating hepatocyte proliferation following acetaminophen-induced tissue injury. Delayed cytokine signaling may account for reduced hepatocyte proliferation and contribute to exaggerated acetaminophen-induced hepatotoxicity in TNFR1-/- mice.

Role of p55 tumor necrosis factor receptor 1 in acetaminophen-induced antioxidant defense.

Tumor necrosis factor (TNF)-alpha is a macrophage-derived proinflammatory cytokine implicated in hepatotoxicity. In the present studies, p55 TNF receptor 1 (TNFR1) -/- mice were used to assess the role of TNF-alpha in acetaminophen-induced antioxidant defense. Treatment of wild-type (WT) mice with acetaminophen (300 mg/kg) resulted in centrilobular hepatic necrosis and increased serum alanine transaminases. This was correlated with a rapid depletion of hepatic glutathione (GSH). Whereas in WT mice GSH levels returned to control after 6-12 h, in TNFR1-/- mice recovery was delayed for 48 h. Delayed induction of heme oxygenase-1 and reduced expression of CuZn superoxide dismutase were also observed in TNFR1-/- compared with WT mice. This was associated with exaggerated hepatotoxicity. In WT mice, acetaminophen caused a time-dependent increase in activator protein-1 nuclear binding activity and in c-Jun expression. This response was significantly attenuated in TNFR1-/- mice. Constitutive NF-kappaB binding activity was detectable in livers of both WT and TNFR1-/- mice. A transient decrease in this activity was observed 3 h after acetaminophen in WT mice, followed by an increase that was maximal after 6-12 h. In contrast, in TNFR1-/- mice, acetaminophen-induced decreases in NF-kappaB activity were prolonged and did not return to control levels for 24 h. These data indicate that TNF-alpha signaling through TNFR1 plays an important role in regulating the expression of antioxidants in this model. Reduced generation of antioxidants may contribute to the increased sensitivity of TNFR1-/- mice to acetaminophen.

Reduced hepatotoxicity of acetaminophen in mice lacking inducible nitric oxide synthase: potential role of tumor necrosis factor-alpha and interleukin-10.

Macrophage-derived inflammatory mediators have been implicated in tissue injury induced by a number of hepatotoxicants. In the present studies, we used transgenic mice with a targeted disruption of the gene for inducible nitric oxide synthase (NOS II) to analyze the role of nitric oxide in inflammatory mediator production in the liver and in tissue injury induced by acetaminophen. Treatment of wild-type mice with acetaminophen (300 mg/kg) resulted in centrilobular hepatic necrosis, which was evident within 3 h and reached a maximum at 18 h. This was correlated with NOS II expression and nitrotyrosine staining of the liver, which was most prominent after 6 h. Expression of mRNA for tumor necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10), matrix metalloproteinase-9, and connective tissue growth factor (CTGF) also increased in the liver following acetaminophen treatment of wild-type mice. NOS II knockout mice were found to be less sensitive to the hepatotoxic effects of acetaminophen than wild-type mice. This did not appear to be due to differences in acetaminophen-induced glutathione depletion or adduct formation. In NOS II knockout mice treated with acetaminophen, hepatic expression of TNF-alpha, as well as CTGF, was significantly increased compared to wild-type mice. In contrast, IL-10 expression was reduced. These data demonstrate that nitric oxide is important in hepatotoxicity induced by acetaminophen. Moreover, some of its effects may be mediated by altering production of pro- and antiinflammatory cytokines and proteins important in tissue repair.

Exaggerated hepatotoxicity of acetaminophen in mice lacking tumor necrosis factor receptor-1. Potential role of inflammatory mediators.

Transgenic mice with a targeted disruption of the tumor necrosis factor receptor 1 (TNFR1) gene were used to analyze the role of TNF-alpha in pro- and anti-inflammatory mediator production and liver injury induced by acetaminophen. Treatment of wild-type mice with acetaminophen (300 mg/kg) resulted in centrilobular hepatic necrosis. This was correlated with expression of inducible nitric oxide synthase (NOS II) and nitrotyrosine staining of the liver. Expression of macrophage chemotactic protein-1 (MCP-1), KC/gro, interleukin-1beta (IL-1beta), matrix metalloproteinase-9 (MMP-9), and connective tissue growth factor (CTGF), inflammatory mediators known to participate in tissue repair, as well as the anti-inflammatory cytokine, interleukin-10 (IL-10), also increased in the liver following acetaminophen administration. TNFR1(-/-) mice were found to be significantly more sensitive to the hepatotoxic effects of acetaminophen than wild-type mice. This was correlated with more rapid and prolonged induction of NOS II in the liver and changes in the pattern of nitrotyrosine staining. Acetaminophen-induced expression of MCP-1, IL-1beta, CTGF, and MMP-9 mRNA was also delayed or reduced in TNFR1(-/-) mice relative to wild-type mice. In contrast, increases in IL-10 were more rapid and more pronounced. These data demonstrate that signaling through TNFR1 is important in inflammatory mediator production and toxicity induced by acetaminophen.