Evaluation of chronic toxicity of cyclocreatine in beagle dogs after oral gavage administration for up to 23 weeks.

Cyclocreatine (LUM-001) was evaluated for chronic toxicity (23 weeks) in beagle dogs to support clinical development in patients with creatine transporter deficiency (CTD) disorder. Deionized water (vehicle control) or cyclocreatine was administered by oral gavage twice daily (12 ± 1 h apart) at 20, 40 and 75 mg/kg/dose followed by a recovery period. Due to severe toxicity, the study was terminated earlier than the planned 39 weeks of dosing. Animals in the 20, 40 and 75 mg/kg/dose groups completed 160, 106, and 55 days of dosing, respectively, followed by 30, 55 and 106 days of a recovery period, respectively. Three (25%), 7 (58%), and 7 (58%) animals were euthanized and/or found dead in the 40, 80, and 150 mg/kg/day dose groups, respectively. Clinical signs observed were inappetence, frequent emesis, stool abnormalities, weight loss, lethargy and respiratory distress. Histopathological evaluation revealed congestion, edema, cellular infiltration, fibrin, and/or hemorrhage in the lungs of all dose groups. Additionally, animals in all cyclocreatine treatment groups had perinuclear cytoplasmic vacuoles in the heart, kidneys, skeletal and smooth muscles. After the recovery period, the vacuoles were still observed in the cardiac and renal tissues. Cyclocreatine was absorbed rapidly with mean Tmax within 1 to 2 h and half-life ranged between 2.17 and 2.79 h on Day 1, however, on the final day of dosing, it ranged between 5.80 and 8.77 h (males) and 10.3 to 13.1 h (females). To conclude, in this study the lungs, kidneys, heart, skeletal and smooth muscles were identified as the target organs of cyclocreatine toxicity in beagle dogs.

Use of an animal model of disease for toxicology enables identification of a juvenile no observed adverse effect level for cyclocreatine in creatine transporter deficiency.

In standard general toxicology studies in two species to support clinical development, cyclocreatine, a creatine analog for the treatment of creatine transporter deficiency, caused deaths, convulsions, and/or multi-organ pathology. The potential translatability of these findings to patients was evaluated by comparing toxicity of cyclocreatine in wild-type mice to creatine transporter-deficient mice, a model of the human disease. A biodistribution study indicated greater accumulation of cyclocreatine in the brains of wild-type mice, consistent with its ability to be transported by the creatine transporter. Subsequent toxicology studies confirmed greater sensitivity of wild-type mice to cyclocreatine-induced toxicity. Exposure at the no observed adverse effect level in creatine transporter-deficient (554 µg*hr/ml) mice exceeded exposure at the maximum tolerated dose in wild-type (248 µg*hr/ml) mice. When dosed at 300 mg/kg/day for 3 months, cyclocreatine-related mortality, convulsions, and multi-organ pathology were observed in wild-type mice whereas there were no adverse findings in creatine transporter-deficient mice. Brain vacuolation was common to both strains. Although transporter-deficient mice appeared to be more sensitive, the finding had no functional correlates in this strain. The results highlight the importance of considering models of disease for toxicology in cases where they may be relevant to assessing safety in the intended patient population.

Serotonergic Innervations of the Orbitofrontal and Medial-prefrontal Cortices are Differentially Involved in Visual Discrimination and Reversal Learning in Rats.

Cross-species studies have identified an evolutionarily conserved role for serotonin in flexible behavior including reversal learning. The aim of the current study was to investigate the contribution of serotonin within the orbitofrontal cortex (OFC) and medial prefrontal cortex (mPFC) to visual discrimination and reversal learning. Male Lister Hooded rats were trained to discriminate between a rewarded (A+) and a nonrewarded (B-) visual stimulus to receive sucrose rewards in touchscreen operant chambers. Serotonin was depleted using surgical infusions of 5,7-dihydroxytryptamine (5,7-DHT), either globally by intracebroventricular (i.c.v.) infusions or locally by microinfusions into the OFC or mPFC. Rats that received i.c.v. infusions of 5,7-DHT before initial training were significantly impaired during both visual discrimination and subsequent reversal learning during which the stimulus-reward contingencies were changed (A- vs. B+). Local serotonin depletion from the OFC impaired reversal learning without affecting initial discrimination. After mPFC depletion, rats were unimpaired during reversal learning but slower to respond at the stimuli during all the stages; the mPFC group was also slower to learn during discrimination than the OFC group. These findings extend our understanding of serotonin in cognitive flexibility by revealing differential effects within two subregions of the prefrontal cortex in visual discrimination and reversal learning.

Evaluation of chronic toxicity of cyclocreatine, a creatine analog, in Sprague Dawley rat after oral gavage administration for up to 26 weeks.

Cyclocreatine (LUM-001), a creatine analog, was evaluated for its nonclinical toxicity in Sprague Dawley (SD) rats. Deionized water as a vehicle control article or cyclocreatine was administered by oral gavage twice daily (approximately 12 ± 1 h apart) at 30, 100 and 300 mg/kg/dose levels in rats up to 26 weeks followed by a 28-day recovery period. Due to an increased incidence of seizures, the 600 mg/kg/day dose group males were dosed only for 16-weeks followed by a 14-week recovery period. Thirteen males and four females from 600 mg/kg/day dose group were sacrificed at interim on Day 113 to study plausible brain lesions and not due to moribundity. There was a dose dependent increase in the number of seizure incidences in ≥60 mg/kg/day males and 600 mg/kg/day females. Microscopically, higher incidences of vacuoles in the brain at 600 mg/kg/day in both sexes, thyroid follicular atrophy and follicular cell hypertrophy at ≥200 mg/kg/day in males and 600 mg/kg/day in females, and seminiferous tubular degeneration and/or interstitial edema in testes at ≥200 mg/kg/day were observed. Mean plasma half-life of cyclocreatine was between 3.5 and 6.5 h. In conclusion, chronic administration of cyclocreatine by oral gavage in Sprague Dawley rats induced the seizures and microscopic lesions in the brain, testes and thyroid. Based on the results of this study the highest tested dose of 600 mg/kg/day (mean Cmax of 151.5 µg/mL; AUC0-24 of 1970 h*µg/mL) was considered the maximum tolerated dose (MTD) in SD rats.

Neonatal serotonin (5-HT) depletion does not affect spatial learning and memory in rats.

BACKGROUND: Extensive previous research has suggested a role for serotonin (5-HT) in learning and memory processes, both in healthy individuals and pathological disorders including depression, autism and schizophrenia, most of which have a developmental onset. Since 5-HT dysfunction in brain development may be involved in disease etiology, the present investigation assessed the effects of neonatal 5-HT depletion on spatial learning and memory in the Morris water maze (MWM). METHODS: Three days old Sprague-Dawley rats were pretreated with desipramine (20 mg/kg) followed by an intraventricular injection of the selective 5-HT neurotoxin 5,7-dihydroxytryptamine (5,7-DHT, 70 µg). Three months later rats were tested in the MWM. RESULTS: Despite a severe and permanent decrease (80-98%) in hippocampal, prefrontal and striatal 5-HT levels, treatment with 5,7-DHT caused no spatial learning and memory impairment. CONCLUSIONS: Limited involvement of chronic 5-HT depletion on learning and memory does not exclude the possibility that this neurotransmitter has an important neuromodulatory role in these functions. Future studies will be needed to identify the nature of the compensatory processes that are able to allow normal proficiency of spatial learning and memory in 5-HT-depleted rats.

Exposición ambiental al mercurio y valores en orina de los habitantes de la comunidad Boca de Yaracuy: ubicada en la costa centro-norte de Venezuela / Enviromental expossure to mercury and urine values in inhabitants from Boca de Yaracuy community in the Venezuela north central coast

El objetivo de esta investigación fue relacionar la concentración de mercurio urinario de 100 residentes de la comunidad Boca de Yaracuy (zona costera del Estado Carabobo-Venezuela) con los niveles mercuriales encontrados en pescado, agua y sedimento provenientes del Caño El Alpargatón. Durante el estudio fueron captados 4 peces, 2 muestras de agua y tres de sedimento y analizadas por absorción atómica. La media de los niveles de mercurio en la orina de los individuos analizados fue de 3,62 ± 1,09 µg/g creatinina, ubicandose por debajo del valor límites establecido por la Agencia para sustancias tóxicas y registro de enfermedades, Conferencia americana de higienistas industriales gubernamentales y la Organización Mundial de la Salud, además de las Gacetas oficiales venezolanas N° 5382 y 5021. En las muestras ambientales provenientes del Caño. El Alpargatón, no se encontraron niveles elevados de mercurio, así como ausencia de intoxicación por mercurio en sus habitantes. Se requieren de nuevas investigaciones que abarquen un mayor número de elementos que faciliten un mejor abordaje de la contaminación mercurial.

The aim study was to relate the concentration of urinary mercury than 100 residents of the Community Boca Yaracuy (Carabobo state coastal-Venezuela) with mercury levels found in fish, water and sediment from the Caño Alpargatón. During the study were caught 4 fish, 2 samples of water and three samples of sedimentand, analyzed by atomic absorption. The average mercury levels in urine of individuals analyzed was 3,62 ± 1.09 mg/g creatinine, ranking below the limits set by the Agency for toxic substance and disease registry, American conference of gubernamental industrial hygienists, the World Health Organization and the Venezuela Official Gazettes N°5382 and 5021. In environmental samples from the Caño Alpargaton not were found elevanted levels of mercury and mercury poisoning absence of its inhabitants. Further investigations are needed to cover a larger number of elements to be a better approach to mercury pollution.

Elimination of endogenous toxin, creatinine from blood plasma depends on albumin conformation: site specific uremic toxicity & impaired drug binding.

Uremic syndrome results from malfunctioning of various organ systems due to the retention of uremic toxins which, under normal conditions, would be excreted into the urine and/or metabolized by the kidneys. The aim of this study was to elucidate the mechanisms underlying the renal elimination of uremic toxin creatinine that accumulate in chronic renal failure. Quantitative investigation of the plausible correlations was performed by spectroscopy, calorimetry, molecular docking and accessibility of surface area. Alkalinization of normal plasma from pH 7.0 to 9.0 modifies the distribution of toxin in the body and therefore may affect both the accumulation and the rate of toxin elimination. The ligand loading of HSA with uremic toxin predicts several key side chain interactions of site I that presumably have the potential to impact the specificity and impaired drug binding. These findings provide useful information for elucidating the complicated mechanism of toxin disposition in renal disease state.

Response disengagement on a spatial self-ordered sequencing task: effects of regionally selective excitotoxic lesions and serotonin depletion within the prefrontal cortex.

Prefrontal cortex (PFC) is critical for self-ordered response sequencing. Patients with frontal lobe damage are impaired on response sequencing tasks, and increased blood flow has been reported in ventrolateral and dorsolateral PFC in subjects performing such tasks. Previously, we have shown that large excitotoxic lesions of the lateral PFC (LPFC) and orbitofrontal cortex FC (OFC), but not global prefrontal dopamine depletion, markedly impaired marmoset performance on a spatial self-ordered sequencing task (SSOST). To determine whether LPFC or OFC was responsible for the previously observed impairments and whether the underlying neural mechanism was modulated by serotonin, the present study compared the effects of selective LPFC and OFC excitotoxic lesions and 5,7-DHT-induced PFC serotonin depletions in marmosets on SSOST performance. Severe and long-lasting impairments in SSOST performance, including robust perseverative responding, followed LPFC but not OFC lesions. The deficit was ameliorated by task manipulations that precluded perseveration. Depletions of serotonin within LPFC and OFC had no effect, despite impairing performance on a visual discrimination reversal task, thus providing further evidence for differential monaminergic regulation of prefrontal function. In the light of the proposed attentional control functions of ventrolateral PFC and the failure of LPFC-lesioned animals to disengage from the immediately preceding response, it is proposed that this deficit may be due to a failure to attend to and register that a response has been made and thus should not be repeated. However, 5-HT does not appear to be implicated in this response inhibitory capacity.

Are urea and creatinine uremic toxins in the rat?

Urea and creatinine are not generally considered to be important uremic toxins despite evidence from dialysis experiments to the contrary, and despite striking elevations of these nitrogenous waste products in uremia. In order to study this problem in acute uremia, we used a new dietary method for prolonging the survival of bilaterally nephrectomized rats. Urea or creatinine were injected on three successive days starting one day after the inception of uremia. Urea or creatinine injections shortened the survival time of acutely uremic rats, and increased the involution of thymus and spleen. The extra urea, but not creatinine, increased the serum osmolality. These data indicate that urea and creatinine are toxic in the acutely uremic rat. Hypertonicity of the serum may contribute to the toxicity of urea. Additional mechanisms of toxicity and additional toxins are not excluded.

Effects of cyclocreatine in rat hepatocarcinogenesis model.

Cyclocreatine (CCr), a substrate analogue of creatine kinase (CK: EC 2.7.3.2.), exhibits anti-tumor activity in vitro and in vivo. We examined the effects of CCr on the hepatocarcinogenesis of F344 rats caused by treatment with diethylnitrosamine (DEN), partial hepatectomy (PH) or 2-acetylaminofluorene (2-AAF). The rats were given a single intraperitoneal injection of 200 mg of DEN per kg in 0.85% NaCl solution at four weeks of age. Two weeks later they were divided into two groups. One group was continuously fed a commercial powder diet containing 0.02% 2-AAF for 12 weeks and the other was continuously fed a commercial powder diet containing 1% CCr plus 0.02% 2-AAF for 12 weeks. A third group of rats as a control was given only a normal powder diet for 12 weeks. All the groups were subjected to a two-thirds partial hepatectomy (PH) at 3 weeks under avertin anesthesia. To elucidate the inhibitory effect of CCr on chemical induced hepatocarcinogenesis, we examined not only the distribution of glutathione-S-transferase placental form (GST-P) a marker used for tumorigenesis, but also the inhibition of the degree of apoptosis. The number (No./cm2) and area (mm2/cm2) of GST-P positive liver foci were significantly lower in the 2-AAF + CCr treated when compared to the group treated with 2-AAF only. Our data suggest that CCr inhibits the degrees of GST-P-positive cells and apoptosis and is active against hepatocarcinogenesis in rat models. This result points out the unique nature of an anticancer agent that inhibits progression of chemically induced hepatocarcinogenesis of rats.

Nucleus reticularis gigantocellularis and nucleus raphe magnus in the brain stem exert opposite effects on behavioral hyperalgesia and spinal Fos protein expression after peripheral inflammation.

Previous findings indicate that the brain stem descending system becomes more active in modulating spinal nociceptive processes during the development of persistent pain. The present study further identified the supraspinal sites that mediate enhanced descending modulation of behavior hyperalgesia and dorsal horn hyperexcitability (as measured by Fos-like immunoreactivity) produced by subcutaneous complete Freund's adjuvant (CFA). Selective chemical lesions were produced in the nucleus raphe magnus (NRM), the nuclei reticularis gigantocellularis (NGC), or the locus coeruleus/subcoeruleus (LC/SC). Compared to vehicle-injected animals with injection of vehicle alone, microinjection of a serotoninergic neurotoxin 5,7-dihydroxytryptamine into the NRM significantly increased thermal hyperalgesia and Fos protein expression in lumbar spinal cord after hindpaw inflammation. In contrast, the selective bilateral destruction of the NGC with a soma-selective excitotoxic neurotoxin, ibotenic acid, led to an attenuation of hyperalgesia and a reduction of inflammation-induced spinal Fos expression. Furthermore, if the NGC lesion was extended to involve the NRM, the behavioral hyperalgesia and CFA-induced Fos expression were similar to that in vehicle-injected rats. Bilateral LC/SC lesions were produced by microinjections of a noradrenergic neurotoxin, DSP-4. There was a significant increase in inflammation-induced spinal Fos expression, especially in the ipsilateral superficial dorsal horn following LC/SC lesions. These results demonstrated that multiple specific brain stem sites are involved in descending modulation of inflammatory hyperalgesia. Both NRM and LC/SC descending pathways are major sources of enhanced inhibitory modulation in inflamed animals. The persistent hyperalgesia and neuronal hyperexcitability may be mediated in part by a descending pain facilitatory system involving NGC. Thus, the intensity of perceived pain and hyperalgesia is fine-tuned by descending pathways. The imbalance of these modulating systems may be one mechanism underlying variability in acute and chronic pain conditions.

Potential uremic toxins modulate energy metabolism of cardiac myocytes in vitro.

In the present study we investigated the direct effects of potential uremic toxins on the energy metabolism of cultured cardiac myocytes. High-energy phosphates were extracted with perchloric acid and determined by high performance liquid chromatography. Energy charge (calculated from the ratio of [ATP], [ADP] and [AMP] was significantly reduced by 20 mM urea and the combination of creatinine (5 mM) plus urea (200 mM). On the other hand, perfusion with culture media containing clinically relevant amounts of urea (20 mM) or creatinine (1 mM) increased the PCr/ATP ratio. This effect was more pronounced after application of an artificial uremic medium (consisting of uremic serum, urea, creatinine and cytokines) or high amounts of creatinine (5 mM) plus urea (200 mM). As contractility of myocytes is reduced due to application of uremic compounds or uremic serum, we attribute changes in contraction frequency or inotropy to dysregulation of calcium availability within the cell. In fact, the cardiodepressive action of uremic serum (2.5%) could be completely reversed by the calcium agonist, Bay K 8644, thus indicating disturbances in myocardial calcium homeostasis in uremia. Altered calcium regulation by uremic toxins might therefore be responsible for the observed changes in myocardial energy metabolism. These results might contribute to the understanding of the pathogenesis of cardiac damage in end-stage renal disease.

Cyclocreatine in cancer chemotherapy.

Cyclocreatine, an analog of creatine, is an efficient substrate for creatine kinase, but its phosphorylated form is a poor phosphate donor in comparison with creatine phosphate. Cyclocreatine was not very cytotoxic upon 24 h of exposure of human SW2 small-cell lung cancer cells to concentrations of up to 5 mM. However, combinations of cyclocreatine (0.5 mM, 24 h) with each of four antitumor alkylating agents, cis-diamminedichloroplatinum(II), melphalan, 4-hydroperoxycyclophosphamide, and carmustine, resulted in additive to greater-than-additive cytotoxicity toward exponentially growing SW2 cells. The greatest levels of synergy were seen at higher concentrations of 4-hydroperoxycyclophosphamide and carmustine as determined by isobologram analysis. In vivo cyclocreatine (0.5 or 1 g/kg) was more effective if given i.v. rather than i.p. The longest tumor-growth delays, up to 10 days, were produced by extended regimens of cyclocreatine. Cyclocreatine was an effective addition to therapy with standard anticancer agents including cis-diamminedichloroplatinum(II), cyclophosphamide, Adriamycin, or 5-fluorouracil. No additional toxicity was observed when 10 days of cyclocreatine treatment was given with full standard-dose regimens of each drug. The resultant increases in tumor-growth delay were 1.7- to 2.4-fold as compared with those obtained for each of the drugs alone. These results indicate that cyclocreatine may be an effective single agent and an effective addition to combination chemotherapy regimens.

[The toxic effect of serotonin in direct and mediated actions on rat embryos]. / Toksicheskii éffekt serotonin pri priamom i oposredovannom deistvii na émbriony krysy.

Embryotoxic effect of serotonin in direct (intra-amniotic administration) and indirect (subcutaneous administration) treatment was studied. In both cases serotonin causes embryo death and different abnormalities in development in small number of embryos. Mechanism of serotonin embryotoxic effect is under discussion.

Comparison of renal effects of creatinine, creatol and methylguanidine in rats with adenine-induced chronic renal failure.

Creatinine (Cr), creatol and methylguanidine (MG), which accumulate in the body with the progress of renal failure after adenine administration, were given separately to rats in order to compare their toxicities. Food containing adenine was given to rats for 20 days to induce renal failure. Then each of the test substances was administered intraperitoneally, and renal function of the rats was determined. The changes in glomerular filtration rate, renal plasma flow and renal blood flow after administration of Cr were only slight in comparison with those of the control group. Creatol or MG administration induced a significant decrease in renal function. In addition, the level of MG in serum, liver and kidney was extraordinarily high in rats given creatol or MG. The toxic effects are discussed on the basis of these results.

Convulsive action and toxicity of uremic guanidino compounds: behavioral assessment and relation to brain concentration in adult mice.

Four guanidino compounds that are known to accumulate in uremia, namely creatinine, guanidine, guanidinosuccinic acid and methylguanidine, were administered intraperitoneally and intracerebroventricularly to adult albino mice and the compounds epileptogenic and toxic properties were behaviorally assessed. After intraperitoneal injection, brain concentration of the compounds as a function of injected dose was monitored additionally. Guanidino compound brain concentration was determined by cation exchange chromatography with fluorescence ninhydrin detection. After systemic administration, especially guanidinosuccinic acid and methylguanidine induced long-lasting generalized convulsions which gradually increased in severity. Increasing the dose injected intraperitoneally resulted in linear increase in brain concentration of the injected compounds, in parallel with increase in proportion of animals presenting with convulsions and/or severity of convulsions. Guanidinosuccinic acid brain concentration increased more slowly than that of the other 3 compounds and guanidinosuccinic acid also exerted its effect later than the others. Since none of the other metabolically related guanidino compounds determined was significantly increased in the brains of the injected animals, the observed behavior was most certainly induced by the compounds injected and not by some secondary metabolite. Epileptogenic properties of the four compounds were markedly and qualitatively different in systemic administration, but rather similar in intracerebral administration. A tentative epileptogenic potency order was inferred from the combined behavioral and biochemical results. All 4 of the compounds tested displayed the ability to induce full-blown clonic-tonic convulsions and they did so in a dose-related manner. Guanidinosuccinic acid appeared to be slightly more potent than methylguanidine, but both guanidinosuccinic acid and methylguanidine were considerably more potent than guanidine. Creatinine was many times less potent than the other 3 guanidino compounds. Revised epileptogenic potency order on the basis of guanidino compound brain concentration after systemic administration as well as potency order after intracerebral administration paralleled the potency order of these compounds in their GABA antagonism reported earlier. It was therefore postulated that the GABA antagonism of uremic guanidino compounds could underlie their epileptogenic character. Moreover, these compounds could very likely be at the basis of the neurological complications including epilepsy of uremic patients in whom they accumulate in physiological fluids and brain.

Effects of creatinine, creatol and methylguanidine on renal function.

Creatinine (Cr), creatol or methylguanidine (MG) was administered intraperitoneally to normal rats, and several renal function parameters were examined in an attempt to clarify the pathology under the various conditions. The glomerular filtration rate, renal plasma flow and renal blood flow after administration of Cr remained virtually unchanged when compared to those in the control group. In contrast, administration of creatol induced a significant decrease in these renal functions. Further decreases in the renal functions were observed in the rats given MG. However, the level of serum MG was extraordinarily high in the rats given creatol or MG. The toxic effects are discussed on the basis of the above results.

Insuficiencia renal cronica avanzada y embarazo: presentacion de 3 casos / Chronic renal failure and pregnancy: a report of 3 cases

Se han publicado numerosos reportes de embarazo en pacientes con isuficiencia renal cronica, con creatinina (Cr) <- 1.5 mg/dl, pero el embarazo es poco comun con niveles de Cr mayores, principalemente >-3 mg/dl. Es probable que los niveles bajos de progesteron o altos de rplactina jueguen un papel clave aqui. Publicamos tres casos con Cr>-6 mg/dl durante el embarazo y postparto. Recomndamos que en toda mujer embarazada o que planee embarazarse se determine la Cr

There are many reports on pregnancy In chronic renal failure with creatinine (Cr) levels ≤ 1.5 mg/dl, but pregnancy is uncommon when the levels are above this limit and specially over 3 mg/dl. It is likely that hormonal abnormalities involving low progesterone and high prolactine levels play a key role. We report on three cases with Cr levels equal to or greater than 6 mg/dl during pregnancy or after delivery. We recommend that Cr levels be determined in the initial follow-up of any woman who wishes to become pregnant.

[The myoelectrical activity of the gastroduodenal area in ulcer formation due to serotonin]. / Mioélektricheskaia aktivnost' gastroduodenal'noi zony pri iazvoobrazovanii, vyzvannom serotoninom.

The effects of prolonged subcutaneous administration of serotonin on the myoelectric activity of the gastroduodenal junction were investigated in conscious rabbits. Serotonin produced the duodenogastric discoordination by increasing duodenal activity and decreasing the activity of stomach and pylorus. This discoordination resulted in gastric ulceration. Atropine prevented both duodenogastric discoordination and ulceration. These results indicate that serotonin ulceration is related to duodenogastric discoordination.