L-citrulline: A preclinical safety biomarker for the small intestine in rats and dogs in repeat dose toxicity studies.

INTRODUCTION: Gastrointestinal (GI) toxicity is still an issue within drug development, especially for novel oncology drugs. The identification of GI mucosal damage at an early stage with high sensitivity and specificity across preclinical species and humans remains difficult. To date, in preclinical studies, no qualified mechanistic, diagnostic or prognostic biomarkers exist for GI mucosal toxicity. L-citrulline is one of the most promising biomarker candidates used in clinical settings to quantify enterocyte integrity in various small intestinal diseases. L-citrulline is an intermediate metabolic amino acid produced mainly by functional enterocytes of the small intestine, whereby enterocyte loss will cause a drop in circulating L-citrulline. METHODS: In several repeat-dose toxicity studies, plasma L-citrulline has been evaluated as a potential safety biomarker for intestinal toxicity in beagle dogs and Wistar (Han) rats treated with different oncological drug candidates in drug development. Clinical observations and body weight determinations were performed during the pretreatment, treatment and treatment-free recovery period as well as toxicokinetic, gross and histopathology examinations. The quantitative determination of plasma L-citrulline levels during the pretreatment (only dogs), treatment and treatment-free recovery period were performed using an HPLC MS/MS assay. In cynomolgus monkeys, the first investigations on baseline L-citrulline levels were performed. RESULTS: In dogs, a dose- and exposure-dependent decrease of up to 50% in plasma L-citrulline was seen without histopathological alterations. However, a decrease of more than 50% in comparison to the individual animal pretreatment value of L-citrulline correlated very well with histopathological findings (intestinal crypt necrosis, villus atrophy, enterocyte loss) and clinical signs (bloody faeces and diarrhoea). During a treatment-free recovery period, a trend of increasing levels was observed in dogs. In rats, absolute L-citrulline plasma levels of treated animals decreased compared to the values of the concurrent control group. This decrease also correlated with the histopathological findings in the small intestine (single cell necrosis and mucosa atrophy). Because of a large physiological variation in L-citrulline plasma levels in dogs and rats, a clear cut-off value for absolute L-citrulline levels predictive of intestinal mucosal toxicity was difficult to establish. However, a > 50% decrease in L-citrulline plasma levels during the treatment period strongly correlated with histopathological findings. DISCUSSION: Based on the performed analysis, a longitudinal investigation of L-citrulline plasma levels for individual animals in the control and treatment groups is essential and pretreatment values of L-citrulline levels in rodents would be highly informative. Overall, further cross-species comparison (Cynomolgus monkey, mouse) and implementation in clinical trials as exploratory biomarker is essential to foster the hypothesis and to understand completely the clinical relevance of L-citrulline as a small intestine biomarker.

l-citrulline: A preclinical safety biomarker for the small intestine in rats and dogs in repeat dose toxicity studies.

INTRODUCTION: Gastrointestinal (GI) toxicity is still an issue within drug development, especially for novel oncology drugs. The identification of GI mucosal damage at an early stage with high sensitivity and specificity across preclinical species and humans remains difficult. To date, in preclinical studies, no qualified mechanistic, diagnostic or prognostic biomarkers exist for GI mucosal toxicity. l-citrulline is one of the most promising biomarker candidates used in clinical settings to quantify enterocyte integrity in various small intestinal diseases. l-citrulline is an intermediate metabolic amino acid produced mainly by functional enterocytes of the small intestine, whereby enterocyte loss will cause a drop in circulating l-citrulline. METHODS: In several repeat-dose toxicity studies, plasma l-citrulline has been evaluated as a potential safety biomarker for intestinal toxicity in beagle dogs and Wistar (Han) rats treated with different oncological drug candidates in drug development. Clinical observations and body weight determinations were performed during the pretreatment, treatment and treatment-free recovery period as well as toxicokinetic, gross and histopathology examinations. The quantitative determination of plasma l-citrulline levels during the pretreatment (only dogs), treatment and treatment-free recovery period were performed using an HPLC MS/MS assay. In cynomolgus monkeys, the first investigations on baseline l-citrulline levels were performed. RESULTS: In dogs, a dose- and exposure-dependent decrease of up to 50% in plasma l-citrulline was seen without histopathological alterations. However, a decrease of more than 50% in comparison to the individual animal pretreatment value of l-citrulline correlated very well with histopathological findings (intestinal crypt necrosis, villus atrophy, enterocyte loss) and clinical signs (bloody faeces and diarrhoea). During a treatment-free recovery period, a trend of increasing levels was observed in dogs. In rats, absolute l-citrulline plasma levels of treated animals decreased compared to the values of the concurrent control group. This decrease also correlated with the histopathological findings in the small intestine (single cell necrosis and mucosa atrophy). Because of a large physiological variation in l-citrulline plasma levels in dogs and rats, a clear cut-off value for absolute l-citrulline levels predictive of intestinal mucosal toxicity was difficult to establish. However, a > 50% decrease in l-citrulline plasma levels during the treatment period strongly correlated with histopathological findings. DISCUSSION: Based on the performed analysis, a longitudinal investigation of l-citrulline plasma levels for individual animals in the control and treatment groups is essential and pretreatment values of l-citrulline levels in rodents would be highly informative. Overall, further cross-species comparison (Cynomolgus monkey, mouse) and implementation in clinical trials as exploratory biomarker is essential to foster the hypothesis and to understand completely the clinical relevance of l-citrulline as a small intestine biomarker.

Protective effect of resveratrol on citrullinemia type I-induced brain oxidative damage in male rats.

Citrullinemia Type I is an inborn error, which leads to accumulation of citrulline and ammonia in blood and body tissues. We evaluated the in vitro effects of citrulline, ammonia and the influence of resveratrol on oxidative stress parameters in the cerebrum of 30- and 60-day-old male Wistar rats. Citrulline (0.1, 2.5, 5.0 mM), ammonia (0.01, 0.1, 1.0 mM) and resveratrol (0.01, 0.1, 0.5 mM) were added to the assays to measure thiobarbituric acid reactive substances (TBA-RS), total sulfhydryl content and the activity of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Citrulline (2.5 and 5.0 mM) increased TBA-RS in the cerebellum of 30-day-old and in the cerebral cortex and cerebellum of 60-day-old. Citrulline (5.0 mM) increased SOD and reduced GSH-Px in the hippocampus of 30-day-old, whereas in the cerebellum it increased GSH-Px. In the cerebral cortex, 2.5 and 5.0 mM citrulline reduced GSH-Px. In 60-day-old, 2.5 and 5.0 mM citrulline increased SOD in the cerebellum, increased GSH-Px in the cerebral cortex and 5.0 mM citrulline reduced CAT and increased SOD in the cerebral cortex. Ammonia (0.1 and 1.0 mM) reduced the sulfhydryl content in the cerebral cortex of 30- and 60-day-old, 1.0 mM ammonia increased SOD and reduced GSH-Px in the cerebellum of 30-day-old and increased SOD in the hippocampus and cerebellum of 60-day-old. Resveratrol was able to prevent the majority of these alterations. Thus, citrulline and ammonia induce oxidative stress in the cerebrum of rats; however, resveratrol was able to exert antioxidant effects against these substances.

Effects of resveratrol on alterations in cerebrum energy metabolism caused by metabolites accumulated in type I citrullinemia in rats.

We investigated the in vitro effects of citrulline (0.1, 2.5 and 5.0 mM) and ammonia (0.01, 0.1 and 1.0 mM), and the influence of resveratrol (0.01 mM, 0.1 mM and 0.5 mM) on pyruvate kinase, citrate synthase, succinate dehydrogenase (SDH), complex II, and cytochrome c oxidase activities in cerebral cortex, cerebellum and hippocampus homogenates of 60-day-old male Wistar rats. Results showed that 2.5 and 5.0 mM citrulline decreased pyruvate kinase activity in cerebral cortex and, at a concentration of 5.0 mM, increased its activity in hippocampus. Additionally, 5.0 mM citrulline increased citrate synthase activity in the cerebellum of rats. Citrulline (5.0 mM) reduced complex II and cytochrome c oxidase activities in cerebral cortex and hippocampus. With regard to ammonia, at 0.1 and 1.0 mM, decreased complex II activity in cerebral cortex and at 1.0 mM decreased its activity in cerebellum and hippocampus. Ammonia (1.0 mM) also decreased cytochrome c oxidase activity in cerebral cortex and cerebellum of rats. Resveratrol was able to prevent most of the alterations caused by these metabolites in the biomarkers of energy metabolism measured in the cerebrum of rats. Data suggest that these alterations in energy metabolism, caused by citrulline and ammonia, are probably mediated by the generation of free radicals, which can in turn be scavenged by resveratrol.

Development of a System of High Ornithine and Citrulline Production by a Plant-Derived Lactic Acid Bacterium, Weissella confusa K-28.

As a bacterium used in industry for production of several amino acids, an endotoxin-free Corynebacterium (C.) glutamicum is well known. However, it is also true that the endotoxin-producing other Corynebacterium species is present. An aim of this study is to obtain a lactic acid bacterium (LAB) that produces ornithine and citrulline at high levels. We successfully isolated a strain, designated K-28, and identified it as Weissella (W.) confusa. The production of ornithine and citrulline by K-28 was 18 ± 1 and 10 ± 2 g/L, respectively, with a 100 ± 9% conversion rate when arginine was continuously fed into a jar fermenter. Although the ornithine high production using C. glutamicum is industrially present, the strains have been genetically modified. In that connection, the wild-type of C. glutamicum produces only 0.5 g/L ornithine, indicating that W. confusa K-28 is superior to C. glutamicum to use a probiotic microorganism. We confirmed that W. confusa K-28 harbors an arginine deiminase (ADI) gene cluster, wkaABDCR. The production of ornithine and the expression of these genes significantly decreased under the aerobic condition rather than anaerobic one. The expression level of the five genes did not differ with or without arginine, suggesting that the production of amino acids in the K-28 strain was not induced by exogenous arginine.

Systemic L-citrulline prevents cerebral vasospasm in haptoglobin 2-2 transgenic mice after subarachnoid hemorrhage.

BACKGROUND: Nitric oxide (NO) depletion and periadventitial inflammation contribute to the pathogenesis of cerebral vasospasm. L-Citrulline increases L-arginine levels, thereby raising NO synthesis. Transgenic C57Bl6 mice with a haptoglobin (Hp) 2-2 genotype develop more severe vasospasm than wild-type (Hp 1-1) mice after subarachnoid hemorrhage (SAH). OBJECTIVE: To evaluate the toxicity of systemic L-citrulline and its effect on basilar artery (BA) vasospasm, neurobehavioral scores, and inducible NO synthase (iNOS)/endothelial NO synthase (eNOS) expression after SAH in Hp 2-2 mice. METHODS: The Hp 2-2 genotypes were confirmed by reverse-transcriptase polymerase chain reaction. Toxicity was assessed with escalating L-citrulline doses. To test efficacy, Hp 1-1 and Hp 2-2 mice (n = 64) were divided into 4 groups (n = 32 per genotype): sham surgery (n = 8), SAH with no treatment (n = 8), SAH + vehicle (n = 8), and SAH + L-citrulline (200 mg/kg IP every 8 hours; n = 8). Post-SAH neurobehavioral scores were recorded at 24 hours; animals were perfused; and BAs were processed for analysis. Expression of iNOS and eNOS was determined by reverse-transcriptase polymerase chain reaction. RESULTS: The administration of L-citrulline resulted in higher BA lumen patencies in both genotypes (Hp 1-1: SAH + vehicle, 77.8 ± 3.2% vs SAH + L-citrulline, 91.8 ± 5.9% [mean ± SEM]; P < .05; Hp 2-2: SAH + vehicle, 67.1 ± 2.0% vs SAH + L-citrulline, 86.9 ± 2.2%; P < .001). Neurobehavioral scores were higher in Hp 2-2 mice treated with L-citrulline (SAH + vehicle, 1.2 ± 0.2 vs SAH + L-citrulline, 2.4 ± 0.2; P < .01). Expression of iNOS and eNOS increased in Hp 2-2 mice after L-citrulline treatment, but limited sample sizes prevented further statistical analysis. L-Citrulline was not toxic even at the highest dose. CONCLUSION: L-Citrulline is safe; increases BA patency, neurobehavioral scores, and NOS expression in Hp 2-2 mice after SAH; and is a potential agent for treatment of vasospasm after SAH.

Effects of nitric oxide on the survival and neuritogenesis of cerebellar Purkinje neurons.

Nitric oxide has been investigated widely both during neurodevelopment and in neurological diseases. However, whilst it has been established that nitric oxide-producing enzymes of nitric oxide synthase family are expressed in cerebellar Purkinje neurons, the effects of nitric oxide on the viability and morphology of these neurons remain unknown. Here, we have demonstrated that the activity of neuronal nitric oxide synthase, but not the inducible or endothelial forms of this enzyme, is required to support the survival of a proportion of cerebellar Purkinje neurons in vitro. We discovered that donation of high concentrations of exogenous nitric oxide reduces Purkinje neuron survival in culture and that peroxynitrite is also toxic to these cells. Finally, we demonstrated that exogenous nitric oxide and peroxynitrite reduce both the magnitude and the complexity of the neurite arbour extended by cerebellar Purkinje neurons. Taken together, these findings reveal that whilst a low level of endogenous nitric oxide, released by the activity of neuronal nitric oxide synthase, is beneficial to cerebellar Purkinje neurons in vitro, high levels of exogenous nitric oxide and peroxynitrite are detrimental to both the survival of these neurons and to their ability to extend processes and form functional neural networks.

Dual mechanism of brain damage induced in vivo by the major metabolites accumulating in hyperornithinemia-hyperammonemia-homocitrullinuria syndrome.

Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is an autosomal recessive disorder caused by a defect in the mitochondrial ornithine transporter, leading to accumulation of ornithine (Orn), homocitrulline (Hcit) and ammonia. Progressive neurological regression whose pathogenesis is not well established is common in this disease. The present work investigated the in vivo effects of intracerebroventricular administration of Orn and Hcit on important parameters of oxidative stress and energy metabolism in cerebral cortex from young rats. Orn and Hcit significantly increased thiobarbituric acid-reactive substances values and carbonyl formation, indicators of lipid and protein oxidative damage, respectively. Furthermore, N-acetylcysteine and the combination of the free radical scavengers ascorbic acid plus α-tocopherol attenuated the lipid oxidation and totally prevented the protein oxidative damage provoked by Orn and Hcit, suggesting that reactive species were involved in these effects. Hcit, but not Orn administration, also decreased glutathione concentrations, as well as the activity of catalase and glutathione peroxidase, indicating that Hcit provokes a reduction of brain antioxidant defenses. As regards to the parameters of energy metabolism, we verified that Orn and Hcit significantly inhibited the citric acid cycle function (inhibition of CO(2) synthesis from [1-(14)C] acetate), the aerobic glycolytic pathway (reduced CO(2) production from [U-(14)C] glucose) and complex I-III activity of the respiratory chain. Hcit also inhibited the activity of aconitase, an enzyme very susceptible to free radical attack. Taken together, our data indicate that mitochondrial homeostasis is disturbed by Orn and especially by Hcit. It is presumed that the impairment of brain bioenergetics and the oxidative damage induced by these metabolites may possibly contribute to the brain deterioration and neurological symptoms affecting patients with HHH syndrome.

A new severe acute necrotizing pancreatitis model induced by L-ornithine in rats.

OBJECTIVE: Intraperitoneal administration of large doses of L-arginine is known to induce severe acute pancreatitis in rats. We therefore set out to determine whether metabolites of L-arginine (L-ornithine, L-citrulline, and nitric oxide) cause pancreatitis. DESIGN: The authors conducted an in vivo animal study. SETTING: This study was conducted at a university research laboratory. SUBJECTS: Study subjects were male Wistar rats. INTERVENTIONS: Dose-response and time course changes of laboratory and histologic parameters of pancreatitis were determined after L-arginine, L-ornithine, L-citrulline, or sodium nitroprusside (nitric oxide donor) injection. MEASUREMENTS AND MAIN RESULTS: Intraperitoneal injection of 3 g/kg L-ornithine but not L-citrulline or nitroprusside caused severe acute pancreatitis; 4 to 6 g/kg L-ornithine killed the animals within hours. Serum and ascitic amylase activities were significantly increased, whereas pancreatic amylase activity was decreased after intraperitoneal injection of 3 g/kg L-ornithine. The increase in pancreatic trypsin activity (9-48 hrs) correlated with the degradation of IkappaB proteins and elevated interleukin-1beta levels. Oxidative stress in the pancreas was evident from 6 hrs; HSP72 synthesis was increased from 4 hrs after L-ornithine administration. Morphologic examination of the pancreas showed massive interstitial edema, apoptosis, and necrosis of acinar cells and infiltration of neutrophil granulocytes and monocytes 18 to 36 hrs after 3 g/kg L-ornithine injection. One month after L-ornithine injection, the pancreas appeared almost normal; the destructed parenchyma was partly replaced by fat. Equimolar administration of L-arginine resulted in lower pancreatic weight/body weight ratio, pancreatic myeloperoxidase activity, and histologic damage compared with the L-ornithine-treated group. L-ornithine levels in the blood were increased 54-fold after intraperitoneal administration of L-arginine. CONCLUSIONS: We have developed a simple, noninvasive model of acute necrotizing pancreatitis in rats by intraperitoneal injection of 3 g/kg L-ornithine. Interestingly, we found that, compared with L-arginine, L-ornithine was even more effective at inducing pancreatitis. Large doses of L-arginine produce a toxic effect on the pancreas, at least in part, through L-ornithine.