Trovafloxacin enhances lipopolysaccharide-stimulated production of tumor necrosis factor-α by macrophages: role of the DNA damage response.

Trovafloxacin (TVX) is a drug that has caused idiosyncratic, drug-induced liver injury (IDILI) in humans. In a murine model of IDILI, otherwise nontoxic doses of TVX and the inflammagen lipopolysaccharide (LPS) interacted to produce pronounced hepatocellular injury. The liver injury depended on a TVX-induced, small but significant prolongation of tumor necrosis factor-α (TNF) appearance in the plasma. The enhancement of TNF expression by TVX was reproduced in vitro in RAW 264.7 murine macrophages (RAW cells) stimulated with LPS. The current study was designed to identify the molecular target of TVX responsible for this response in RAW cells. An in silico analysis suggested a favorable binding profile of TVX to eukaryotic topoisomerase II-α (TopIIα), and a cell-free assay revealed that TVX inhibited eukaryotic TopIIα activity. Topoisomerase inhibition is known to lead to DNA damage, and TVX increased the DNA damage marker phosphorylated histone 2A.X in RAW cells. Moreover, TVX induced activation of the DNA damage sensor kinases, ataxia telangiectasia mutated (ATM) and Rad3-related (ATR). The ATR inhibitor NU6027 [6-(cyclohexylmethoxy)-5-nitrosopyrimidine-2,4-diamine] prevented the TVX-mediated increases in LPS-induced TNF mRNA and protein release, whereas a selective ATM inhibitor [2-(4-morpholinyl)-6-(1-thianthrenyl)-4H-pyran-4-one (KU55933)] was without effect. TVX prolonged TNF mRNA stability, and this effect was largely attenuated by NU6027. These results suggest that TVX can inhibit eukaryotic topoisomerase, leading to activation of ATR and potentiation of TNF release by macrophages, at least in part through increased mRNA stability. This off-target effect might contribute to the ability of TVX to precipitate IDILI in humans.

Comparison of TNFα to lipopolysaccharide as an inflammagen to characterize the idiosyncratic hepatotoxicity potential of drugs: Trovafloxacin as an example.

Idiosyncratic drug reactions (IDRs) are poorly understood, unpredictable, and not detected in preclinical studies. Although the cause of these reactions is likely multi-factorial, one hypothesis is that an underlying inflammatory state lowers the tolerance to a xenobiotic. Previously used in an inflammation IDR model, bacterial lipopolysaccharide (LPS) is heterogeneous in nature, making development of standardized testing protocols difficult. Here, the use of rat tumor necrosis factor-α (TNFα) to replace LPS as an inflammatory stimulus was investigated. Sprague-Dawley rats were treated with separate preparations of LPS or TNFα, and hepatic transcriptomic effects were compared. TNFα showed enhanced consistency at the transcriptomic level compared to LPS. TNFα and LPS regulated similar biochemical pathways, although LPS was associated with more robust inflammatory signaling than TNFα. Rats were then codosed with TNFα and trovafloxacin (TVX), an IDR-associated drug, and evaluated by liver histopathology, clinical chemistry, and gene expression analysis. TNFα/TVX induced unique gene expression changes that clustered separately from TNFα/levofloxacin, a drug not associated with IDRs. TNFα/TVX cotreatment led to autoinduction of TNFα resulting in potentiation of underlying gene expression stress signals. Comparison of TNFα/TVX and LPS/TVX gene expression profiles revealed similarities in the regulation of biochemical pathways. In conclusion, TNFα could be used in lieu of LPS as an inflammatory stimulus in this model of IDRs.

Tumor necrosis factor alpha is a proximal mediator of synergistic hepatotoxicity from trovafloxacin/lipopolysaccharide coexposure.

The use of trovafloxacin (TVX), a fluoroquinolone antibiotic, was severely restricted because of an association of TVX therapy with idiosyncratic hepatotoxicity in patients. The mechanisms underlying idiosyncratic toxicity are unknown; however, one hypothesis is that an inflammatory stress can render an individual sensitive to the drug. Previously, we reported that treatment of mice with TVX and lipopolysaccharide (LPS) induced tumor necrosis factor (TNF) alpha-dependent liver injury, whereas TVX or LPS treatment alone was nontoxic. The goal of this study was to elucidate the role of TNFalpha in TVX/LPS-induced liver injury. TNF receptor (TNFR) 1 p55(-/-) and TNFR2 (p75(-/-)) mice were protected from hepatotoxicity caused by TVX/LPS coexposure, suggesting that TVX/LPS-induced liver injury requires both TNF receptors. TNFalpha inhibition using etanercept significantly reduced the TVX/LPS-induced increases in the plasma concentrations of several cytokines around the time of onset of liver injury. However, despite the reduction in chemokines, etanercept treatment did not affect the TVX/LPS-induced hepatic accumulation of neutrophils. In addition, etanercept treatment attenuated TVX/LPS induction of plasminogen activator inhibitor-1, and this was associated with a reduction in hepatic fibrin deposition. Mice treated with TVX and a nontoxic dose of TNFalpha also developed liver injury. In summary, TNFalpha acts through p55 and p75 receptors to precipitate an innocuous inflammatory cascade. TVX enhances this cascade, converting it into one that results in hepatocellular injury.

In vitro anti-inflammatory effects and immunomodulation by gemifloxacin in stimulated human THP-1 monocytes.

Cultured human THP-1 monocytes were exposed to serial concentrations of gemifloxacin over 4 h after pre-stimulation with zymogen A for 1 h or Staphylococcus aureus for 2 h. The following parameters were assessed: pH, phagocytosis, c-AMP, NO, TNFalpha, IL-1, IL-6, IL-8 and H2O2 levels, enzyme activities of protein kinase C, NADPH oxidase, SOD, gluthathion reductase, NAG and cathepsin D as well as lipid peroxidation. The reversiblity of these changes was determined in the presence of known blockers of the phagocytic process. The effects of gemifloxacin on DNA synthesis and killing of S. aureus was assessed in bacteria alone and in those bacteria phagocytosed by THP-1 monocytes over 24 h. Gemifloxacin in stimulated THP-1 monocytes over the first 30 min caused an increase in c-AMP, NO, H2O2 and TNFalpha levels and protein kinase C, NADPH oxidase, glutathione reductase, NAG and cathepsin D activities. The pH became more acidic and phagocytosis was stimulated. These parameters were reversed at 1 h and continued to decline until 4 h. Lipid peroxidation was at the highest levels at 1 h and IL-8 levels at 2 h. DNA synthesis and bacterial growth were suppressed at 2 h in both S. aureus alone and bacteria phagocytosed by THP-1 monocytes. These effects were at a higher magnitude at 24 h. Gemifloxacin initiates a phagocyticidal effect of THP-1 monocytes at an early time of 30 min which plays a role in killing bacteria but a higher magnitude of killing of bacteria occurs later by a standard static mechanism. This early action of gemifloxacin should decrease the spread of infection and the inflammatory response since the tissue destruction process was attenuated at 4 h.

Locomotor stereotypy is produced by methylphenidate and amfonelic acid and reduced by haloperidol but not clozapine or thioridazine.

In addition to its well-known behavioral effects in rats, amphetamine also produces patterned locomotion (referred to below as locomotor stereotypy) in an open field. Locomotor stereotypy may be mediated by different mechanisms than those mediating the better-known behavioral effects of amphetamine. To determine whether the ability to produce locomotor stereotypy is an exclusive property of amphetamine or is a property of many amphetamine-like stimulants, several doses of methylphenidate and amfonelic acid were tested. The ability of both atypical and typical neuroleptics to block amphetamine-induced locomotor stereotypy was also tested. Both amfonelic acid and methylphenidate produced some degree of locomotor stereotypy. In addition, amphetamine-induced locomotor stereotypy was reduced by haloperidol but not by clozapine or thioridazine. These data suggest that locomotor stereotypy is more closely related to focused stereotypy than to hyperlocomotion.

Effects of DN-2327, a new anxiolytic, diazepam and buspirone on exploratory activity of the rat in an elevated plus-maze.

In the present study, the effects of a new anxiolytic, DN-2327, were compared to those of diazepam and buspirone in rats in the elevated plus-maze test. Two indices of anxiety were obtained in this test: the number of entries into the open arms expressed as a percentage of the total number of arm entries and the percentage of time spent on the open arms. Both a typical anxiolytic, diazepam, at 2.5, 5 and 10 mg/kg, PO and a new anxiolytic, DN-2327, at 2.5 and 5 mg/kg, PO dose-dependently increased the two indices: the percentage of time spent on the open arms and the percentage of open-arm entries. On the other hand, pentylenetetrazol (PTZ) at 10 and 20 mg/kg, IP decreased the two indices dose dependently as did yohimbine at 1.5 and 3 mg/kg, IP. DN-2327 at 2.5 and 5 mg/kg, PO and diazepam at 5 and 10 mg/kg, PO dose dependently and significantly increased the two indices that were suppressed following administration of PTZ at 10 mg/kg, IP. The effects of both DN-2327, 5 mg/kg, PO, and diazepam, 10 mg/kg, PO, on the two indices were significantly antagonized by the benzodiazepine (BZD) receptor antagonist flumazenil, 20 mg/kg, IP. Buspirone (2.5-20 mg/kg, PO) did not affect either of the two responses but dose dependently decreased the number of rearings, although in the Vogel conflict test, the anti-conflict activity of buspirone was equipotent to that of diazepam and DN-2327 at the minimum effective dose (10 mg/kg, PO) of each drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Suitability of amfonelic acid-induced locomotor stimulation in mice as a model for the evaluation of classical and atypical antipsychotics.

The potential usefulness of amfonelic acid ( AFA ), a selective dopamine (DA)-releasing agent, in quantitatively assessing the antidopaminergic and antipsychotic potencies of drugs, was evaluated. The procedure consisted of determining the ED50S of a number of neuroleptics in inhibiting the locomotor-stimulant effect of amfonelic acid in mice. These results were compared with the published data on the relative potencies of the neuroleptics to induce catalepsy in rats, to block drug-induced stereotypy and to alleviate psychotic symptoms clinically. It was observed that the amfonelic acid model was as good as, but not superior to, the other three procedures in identifying the potencies of classical antipsychotics. This model, however, was able to predict the clinical effectiveness of two atypical antipsychotics, thioridazine and clozapine, much more accurately than could be achieved by the other methods. Certain other atypical antipsychotics such as, mezilamine , RMI 81, 582, sulpiride and sultopride also produced a dose-related blockade of the amfonelic acid induced locomotor stimulation in mice. The antagonism to amfonelic acid exhibited by mezilamine was weaker, and that of RMI 81,582 was stronger than that of chlorpromazine. Only large doses of the two benzamides were effective in blocking the effect of amfonelic acid, sultopride being about 3 times more effective than sulpiride in this regard. Another analogue of benzamide, YM-09151-2, known to have the profile of a classical antipsychotic, was more effective than haloperidol in blocking the stimulant effect of amfonelic acid. Trebenzomine , which is considered to have the properties of an atypical antipsychotic, although this was proved otherwise when tested clinically, actually potentiated the response of mice to amfonelic acid. Apomorphine antagonized the stimulant effect of amfonelic acid, which could be attributed to its agonist activity at presynaptic DA receptors. Apomorphine has been reported to have clinical antipsychotic effects. Certain non-antipsychotic drugs such as prazosin (but not phenoxybenzamine), promethazine, methysergide, diazepam, as well as the gamm -aminobutyric acid agonists, muscimol and THIP, also inhibited the amfonelic acid-induced locomotor stimulation. In spite of this drawback, the present procedure should prove to be a useful animal model for the evaluation of the antipsychotic potencies of drugs. Its ability to identify the potential usefulness of atypical antipsychotics is noteworthy.