Activation of voltage-gated KCNQ/Kv7 channels by anticonvulsant retigabine attenuates mechanical allodynia of inflammatory temporomandibular joint in rats.

BACKGROUND: Temporomandibular disorders (TMDs) are characterized by persistent orofacial pain and have diverse etiologic factors that are not well understood. It is thought that central sensitization leads to neuronal hyperexcitability and contributes to hyperalgesia and spontaneous pain. Nonsteroidal anti-inflammatory drugs (NSAIDs) are currently the first choice of drug to relieve TMD pain. NSAIDS were shown to exhibit anticonvulsant properties and suppress cortical neuron activities by enhancing neuronal voltage-gated potassium KCNQ/Kv7 channels (M-current), suggesting that specific activation of M-current might be beneficial for TMD pain. RESULTS: In this study, we selected a new anticonvulsant drug retigabine that specifically activates M-current, and investigated the effect of retigabine on inflammation of the temporomandibular joint (TMJ) induced by complete Freund's adjuvant (CFA) in rats. The results show that the head withdrawal threshold for escape from mechanical stimulation applied to facial skin over the TMJ in inflamed rats was significantly lower than that in control rats. Administration of centrally acting M-channel opener retigabine (2.5 and 7.5 mg/kg) can dose-dependently raise the head withdrawal threshold of mechanical allodynia, and this analgesic effect can be reversed by the specific KCNQ channel blocker XE991 (3 mg/kg). Food intake is known to be negatively associated with TMJ inflammation. Food intake was increased significantly by the administration of retigabine (2.5 and 7.5 mg/kg), and this effect was reversed by XE991 (3 mg/kg). Furthermore, intracerebralventricular injection of retigabine further confirmed the analgesic effect of central retigabine on inflammatory TMJ. CONCLUSIONS: Our findings indicate that central sensitization is involved in inflammatory TMJ pain and pharmacological intervention for controlling central hyperexcitability by activation of neuronal KCNQ/M-channels may have therapeutic potential for TMDs.

The effect of tribendimidine and its metabolites against Necator americanus in golden hamsters and Nippostrongylus braziliensis in rats.

The antinematode effect of tribendimidine (TBD) and its metabolites has been studied. A total of 107 hamsters were each infected with 250 Necator americanus third stage infective larvae (NaL3) for 25 days. In the first test, 75 hamsters were divided equally into 15 groups for determination of ED(50) and ED(90.) Among them, five groups were treated orally with TBD or its metabolite, p-(1-dimethylamino ethylimino)aniline (aminoamidine, deacylated amidantel, BAY d 9216, dADT), at single doses of 1, 2, 4, 8, and 16 mg/kg. The remaining five groups were administered with acetylated dADT (AdADT) at single oral doses of 8, 12, 18, 24, and 30 mg/kg. In the second test, 20 hamsters were equally divided into four groups. Two groups were treated intramuscularly with TBD and dADT at a single dose of 16 mg/kg, while in the remaining two groups, single intramuscular dose of AdADT 15 or 30 mg/kg was administered. In the third test, two groups of six hamsters were treated orally with terephthalaldehyde (TPAL) and terephthalic acid (TPAC) at a single dose of 1,000 mg/kg. Other 85 rats, each infected with 300 Nippostrongylus braziliensis third stage infective larvae (NbL3), were used in three tests. For determination of ED(50) and ED(90) in the first test, five groups of five rats were treated orally with TBD or dADT at single doses of 3.0, 4.2, 5.9, 8.2, and 11.5 mg/kg or 2.0, 2.9, 4.2, 6.1, and 8.8 mg/kg, respectively. In the second test, three groups of eight to nine rats were treated orally with TBD at a single 8.4-mg/kg dose (ED(90)) and AdADT 100 or 200 mg/kg, respectively. In the third test, two groups of four rats were treated orally with TPAL and TPAC at a single dose of 1,000 mg/kg. Twenty-four to 48 h post-treatment, all the feces of each hamster and rat were collected for recovery of worms expelled from the feces. Following this period, all of the animals were sacrificed, and the adult hookworm or N. braziliensis from small intestine and large intestine were recovered and counted for calculation of worm burden reduction. The results showed that the ED(50) and ED(90) for TBD, dADT, and AdADT determined in treatment of N. americanus-infected hamsters were 1.849 and 13.598, 3.922 and 54.354, as well as 20.966 and 51.633 mg/kg, respectively. In intramuscular administration of TBD and dADT at single dose of 16 mg/kg or AdADT 30 mg/kg, similar worm burden reductions of 71.4-76.3% were observed. Two other metabolites, i.e., TPAL and TPAC, exhibited no effect against N. americanus. The ED(50) and ED(90) for TBD and dADT determined in treatment of rats infected with N. braziliensis were 3.234 and 8.435, as well as 2.345 and 5.104 mg/kg. Oral administration of AdADT at a higher single dose of 100 or 200 mg/kg resulted in worm burden reductions of 11.9-46.3%, which was significantly lower than 84.5% of worm burden reduction obtained from rats treated with TBD 8.4 mg/kg. The results indicate that in oral administration, TBD exhibits slightly better effect against N. americanus in hamsters than dADT, but AdADT possesses less effect; TBD, dADT, and AdADT show promising effect in intramuscular treatment of N. americanus-infected hamsters; the effect of oral dADT against N. braziliensis in rats is somewhat better than TBD, while AdADT endorses poor effect; and TPAL and TPAC are ineffective metabolites of TBD against both species of nematodes.

The in vitro and in vivo effect of tribendimidine and its metabolites against Clonorchis sinensis.

The purpose of the study was to understand the in vitro and in vivo effect of tribendimidine (TBD) and its metabolites of p-(1-dimethylamino ethylimino)aniline (aminoamidine, deacylated amidantel, BAY d 9216, dADT), acetylated dADT (AdADT), terephthalaldehyde (TPAL), and terephthalic acid (TPAC) against adult Clonorchis sinensis. In in vitro test, the adults of C. sinensis were placed to each of the 24 wells of a Falcon plate and maintained in Hanks' balanced salt solution-20% calf serum. Besides observation on the direct in vitro effect of TBD and its metabolites, the worms exposed to TBD and its metabolites for 1-24 h were transferred to the medium without drug and incubated continually for another 72 h. The reversible effect of TBD and its metabolites was assessed by the recovery of worm motor activity and parasite survival. In in vivo test, 235 rats were divided into five batches for oral infection of each rat with 50 C. sinensis metacercariae. Five to 6 weeks post-infection, groups of rats were treated orally or intramuscularly with a single dose of TBD or its metabolites, while untreated but infected rats served as control. All treated rats were killed 2 weeks post-treatment for assessment of efficacy. When adult C. sinensis were exposed to TBD or dADT 0.5 microg/mL, they were paralyzed rapidly accompanied by dilatation of the gut. The in vitro effect of AdADT decreased significantly, which was at least lower than 20- to 40-fold compared with TBD and dADT. TPAL and TPAC at a high concentration of 100 microg/mL exhibited no effect against adult C. sinensis. In the worms exposed to TBD or dADT 1 microg/mL for 1 h, well recovery of the worm motor activity from paralysis was seen in the medium without drug. If exposure time extended to 4-24 h before transferred to the medium without drug, few worms were dead and most worms showed very poor recovery of their activity. When the worms exposed to TBD or dADT 10 microg/mL for 1, 4, and 24 h were transferred to the drug-free medium, recovery of poor motor activity of worms or worm death was seen. In the worms exposed to AdADT 20 and 40 microg/mL for 1-24 h, more worms recovered poor motor activity in the medium without drug. In rats infected with C. sinensis and treated orally with TBD or dADT, the ED(50) and ED(95) were 20.318 and 195.358 mg/kg or 18.969 and 268.882 mg/kg. Under the equal dosages used in the treatment of rats infected with C sinensis, the effects between TBD and dADT or TBD and AdADT were similar. Intramuscular TBD or dADT at a single dose of 12.5-75 mg/kg showed effect against adult C. sinensis harbored in rats. TPAL and TPAC exhibit no effect against C sinensis harbored in rats treated orally with a higher dose of 1 g/kg. The results indicate that TBD and dADT exhibit a strong in vitro effect to paralyze the adult C. sinensis, but less in vitro effect was seen in AdADT. TBD, dADT, and AdADT exhibit similar therapeutic effect in oral treatment of rats infected with C. sinensis, and intramuscular TBD and dADT also show promising effect against C. sinensis in rats. TPAL and TPAC are ineffective metabolites of TBD.

Tribendimidine and albendazole for treating soil-transmitted helminths, Strongyloides stercoralis and Taenia spp.: open-label randomized trial.

BACKGROUND: Tribendimidine is an anthelminthic drug with a broad spectrum of activity. In 2004 the drug was approved by Chinese authorities for human use. The efficacy of tribendimidine against soil-transmitted helminths (Ascaris lumbricoides, hookworm, and Trichuris trichiura) has been established, and new laboratory investigations point to activity against cestodes and Strongyloides ratti. METHODOLOGY/PRINCIPAL FINDINGS: In an open-label randomized trial, the safety and efficacy of a single oral dose of albendazole or tribendimidine (both drugs administered at 200 mg for 5- to 14-year-old children, and 400 mg for individuals > or = 15 years) against soil-transmitted helminths, Strongyloides stercoralis, and Taenia spp. were assessed in a village in Yunnan province, People's Republic of China. The analysis was on a per-protocol basis and the trial is registered with controlled-trials.com (number ISRCTN01779485). Both albendazole and tribendimidine were highly efficacious against A. lumbricoides and, moderately, against hookworm. The efficacy against T. trichiura was low. Among 57 individuals who received tribendimidine, the prevalence of S. stercoralis was reduced from 19.3% to 8.8% (observed cure rate 54.5%, p = 0.107), and that of Taenia spp. from 26.3% to 8.8% (observed cure rate 66.7%, p = 0.014). Similar prevalence reductions were noted among the 66 albendazole recipients. Taking into account "new" infections discovered at treatment evaluation, which were most likely missed pre-treatment due to the lack of sensitivity of available diagnostic approaches, the difference between the drug-specific net Taenia spp. cure rates was highly significant in favor of tribendimidine (p = 0.001). No significant adverse events of either drug were observed. CONCLUSIONS/SIGNIFICANCE: Our results suggest that single-dose oral tribendimidine can be employed in settings with extensive intestinal polyparasitism, and its efficacy against A. lumbricoides and hookworm was confirmed. The promising results obtained with tribendimidine against S. stercoralis and Taenia spp. warrant further investigations. In a next step, multiple-dose schedules should be evaluated.

Assessment of chronic toxicity and carcinogenicity in rats of Wingstay 100, a rubber antioxidant/antiozonant.

The chronic toxicity and carcinogenicity of Wingstay 100 (W 100), a rubber antioxidant/antiozonant, were studied in Fischer 344 (F 344) rats in two chronic studies. Earlier genetic studies indicated that the product had weak activity in a bacterial mutation assay, but lacked activity in chromosomal aberration assays. In an one year study, both genders of F 344 rats were exposed to 53, 310 or 1900 ppm in NIH-07 diet for 52 weeks, and sacrifices were made at 38, 52 and 64 weeks. No test substance related deaths occurred, although the high dose of 1900 ppm caused a decrease in body weight gain and food consumption in both genders. Red blood cell mean corpuscular volume was significantly increased at 38 weeks, accompanied by a significant decrease in mean corpuscular hemoglobin concentration. At 52 weeks, the red blood cell count and hemoglobin values were also significantly decreased in high dose animals of both genders. Total bilirubin and cholesterol were increased in high dose animals of 38 and 52-week sacrifices. During the 3 month recovery, hematology parameters, bilirubin and cholesterol returned to control values. Total protein was reduced in high dose animals of both genders, throughout the entire exposure and recovery periods. W 100 also produced increases in relative liver, spleen, heart and kidney weights in high dose animals. Both genders of all W 100 groups exhibited significant increases in urothelial cell proliferation (measured by PCNA) and adaptive hyperplasia. No regenerative hyperplasia, preneoplasia, or neoplasia were present. There was microscopic evidence of extramedullary erythropoiesis in the spleen and liver of high dose animals in both genders, otherwise no other pertinent microscopic finding was evident. In parallel, an accelerated bioassay (ABA) was conducted, which is a mechanistic initiation/promotion carcinogenicity study designed to assess tumor induction and promotion potential of a test substance in major organs of carcinogenesis. The present study was conducted in male F 344 rats for 38 weeks. The target sites chosen for the ABA were liver and urinary bladder and the dose for W 100 was 1900 ppm previously established to be a toxic dose. The liver tumor initiator was diethylnitrosamine (DEN), and the urinary bladder initiator was N-butyl N-(4-hydroxybutyl) nitrosamine (BBN). The initiators were administered during the first 14 weeks followed by the promoters. The promoters, phenobarbital (PB) for the liver and nitrilotriacetate (NTA) for the urinary bladder, were administered during the last 24 weeks of the study after the test substance. The study had 11 test groups including a negative control. The critical comparisons for initiating activity were conducted between groups 3 (PB) and 6 (W 100 + PB) for the liver and groups 8 (NTA) and 11 (W 100 + NTA) for the urinary bladder. The critical comparisons for promoting activity were conducted between groups 2 (DEN) and 5 (DEN + W 100) for the liver and groups 7 (BBN) and 10 (BBN + W 100) for the urinary bladder. There were 26 and 38-week sacrifices. In this study, most body weight reductions were due to DEN. At 26 weeks, significant increases in liver weights were present in all PB-exposed groups. Significant increases in renal weights occurred in all NTA, BBN and DEN groups. A similar organ weight pattern was present at 38 weeks. At 26 weeks, there were hepatocellular (33%) and urothelial (67%) tumors present in positive control groups (DEN/DEN + PB/BBN/BBN + NTA). In contrast, in the DEN + W 100 (5) and the BBN + W 100 (10) groups no tumors were present indicating absence of promotion. In addition, no tumors were present in groups 6 (W 100 + PB) or 11 (W 100 + NTA) indicating absence of initiation. At 38 weeks, the incidences of hepatocellular adenomas and carcinomas in positive control group (DEN) was 44%. The incidence of urothelial adenomas and carcinomas was 67% in group 7 (BBN). In contrast, groups 5 (DEN + W 100) or group 10 (BBN + W 100) had

Sensitization of mice to paraphenylenediamine and structurally-related compounds: adjuvant effects of vitamin A supplementation.

Allergic contact dermatitis from moderate and weak contact sensitizers is generally studied with guinea pigs, since they are readily sensitized to contact allergens. Mice, by contrast, are poor responders to weak contact allergens. However, the variety of in vitro murine systems as well as murine specific reagents make mice the preferable species. With the use of vitamin A supplementation, 2 protocols were developed which sensitized CBA/J female mice to paraphenylenediamine. Mice were sensitized by 5 daily topical applications to shaven dorsal skin. Alternately, mice were sensitized by 2 intraperitoneal injections of antigen pulsed spleen cells. Sensitization to paraphenylenediamine was determined by ear swelling following topical application. Vitamin A supplementation was found to be essential for optimum response. Lymph node and spleen cells from sensitized mice were capable of proliferating to paraphenylenediamine in vitro. With the use of vitamin A supplementation and intraperitoneal injection, CBA/J mice were also sensitized to a number of compounds structurally related to paraphenylenediamine, including the ortho- and meta-derivatives of paraphenylenediamine, as well as hydroquinone and resorcinol. These new protocols, combined with vitamin A supplementation, expand the use of mice to study moderate sensitizers with minimal animal utilization.

Protection against murine cerebral malaria by dietary-induced oxidative stress.

Feeding 20% (w/w) menhaden-fish oil in a standard laboratory chow diet for 4 wk partially protected CBA/CaJ mice from the central nervous system consequences of infection with Plasmodium berghei (ANKA). Full protection (complete survival for 14 days postinfection) could be obtained by feeding a purified pro-oxidant vitamin E-deficient diet containing 4% (w/w) menhaden oil (MO - VE diet). The purified pro-oxidant MO - VE diet also exerted a pronounced suppressive effect against the parasite (depressed 6-day parasitemias). The anitmalarial effect of the MO - VE diet could be prevented by supplementing the diet with vitamin E or with either of 2 synthetic antioxidants, N,N'-diphenyl-p-phenylenediamine or probucol. These results suggest that the fish oil exerts its antimalarial effect by imposing a dietary-induced oxidative stress on the infected host erythrocyte, the parasite, or both. Nutritional manipulation of host oxidative stress status may be a useful adjunct therapy in patients undergoing treatment with pro-oxidant antimalarials such as drugs of the qinghaosu family.

Dinaline: a new oral drug against leukemia? Preclinical studies in a relevant rat model for human acute myelocytic leukemia (BNML).

The efficacy and toxicity of Dinaline (GOE 1734; PD 104 208; NSC 328786; 4-amino-N-(2'-aminophenyl)benzamide) was evaluated in the Brown Norway acute myelocytic leukemia, which is generally accepted as a relevant preclinical model for human acute myelocytic leukemia. Upon repeated daily oral administration at least an 8 log leukemic cell kill was achieved with only less than a 1 log kill for normal pluripotent hemopoietic stem cells. Daily split-dose treatment even proved to be more effective and resulted in 40-50% cures. However, toxicity was also more pronounced in particular in regard to the gastrointestinal tract. So far, the mode of action of Dinaline is unknown, but its striking therapeutic index warrants further clinical investigation.

Detection of mutagens in the urine of rats following topical application of hair dyes.

Mutagens were detected in the urine of rats following topical application of two commercial oxidative-type hair dye preparations. The test system used was induction of back mutation with the bacterial tester strain TA1538, a histidine-dependent mutant of Salmonella typhimurium. Various quantities of dye were applied to the shortened hair on the backs of the test animals. The dye was allowed to remain on the hair for 20 min after application and was then removed by shampooing and thorough rinsing. Maximal levels of mutagenic activity occurred with urine collected during first 24 h following dye application, and a dose--response was observed when increasing volumes of mutagenic urine were tested. Mutagens were detected in rat urine after intraperitoneal injection, and also after topical application of 4-nitro-o-phenylenediamine, one of the constituents of the hair-dye preparations.

Oral absorption and selective tissue localization of 4'-(9-acridinylamino)-methanesulfon-m-anisidide.

The disposition of 4'-(9-acridinylamino)-methanesulfon-m-anisidide (AMSA), a new antitumor agent presently undergoing clinical evaluation, was studied in mice and rats following oral administration and compared to that observed following intravenous administration. The metabolic fate of AMSA was the same with either intravenous or oral administration; however, the tissue distribution of AMSA differed significantly between the two routes of administration. Following absorption from the GI tract, AMSA was rapidly cleared from plasma by the liver and excreted in the bile as metabolites. Concentrations of AMSA in the liver were relatively high after oral administration and were sufficient to exert a cytotoxic effect on L1210 cells implanted at the site. The results indicate the use of AMSA orally to attain selective localization in the liver with decreased systemic exposure, which may prove useful against tumor metastases to the liver or primary hepatocellular carcinoma.