Antinociceptive action of FK506 in mice

Immunophilins are abundantly present in the brain as compared to the immune system. Immunophilin-binding agents like FK506 are known to inactivate neuronal nitric oxide synthase (nNOS) by inhibiting calcineurin and decrease the production of nitric oxide. Nitric oxide is involved in the mediation of nociception at the spinal level. In the present study, the effect of FK506 on the tail flick response in mice and the possible involvement of NO-L-arginine pathway in this paradigm was evaluated. FK506 (0.5, 1 and 3 mg/kg, ip) produced a significant antinociception in the tail flick test. Nitric oxide synthase (NOS) inhibitor L-NAME significantly and dose dependently (10-40 mg/kg, ip) potentiated the FK506 (0.5 mg/kg)-induced antinociception. On the other hand, NOS substrate L-arginine (100, 200 and 400 mg/kg) inhibited the FK506-induced antinociception in a dose-dependent manner. Concomitant administration of L-NAME (20 and 40 mg/kg) with L-arginine (200 mg/kg) blocked the inhibition exerted by L-arginine on the FK506-induced antinociception. Thus, it was concluded that NO- L-arginine pathway may be involved in the FK506-induced antinociception in tail flick test.     

Effects of Catechol-O-Methyltransferase Inhibitors and L-3,4-Dihydroxyphenylalanine With or Without Carbidopa on Extracellular Dopamine in Rat Striatum

Abstract: The effects of two new catechol- O -methyltransferase (COMT) inhibitors, OR-611 and Ro 40-7592, in combination with L-3,4-dihydroxyphenylalanine (L-dopa) with or without carbidopa on extracellular levels of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 3- O -methyldopa (3-OMD), and 5-hydroxyindoleacetic acid in rat striatum were studied. A dose of 10 mg/kg i.p. of Ro 40-7592 alone, in contrast to the same dose of OR-611, decreased the dialysate level of HVA and increased that of DOPAC; this dose was thus used to differentiate between the effects of central and peripheral COMT inhibition. L-Dopa (50 mg/kg i.p.) alone slightly increased extracellular levels of DA, DOPAC, and HVA. The effects of L-dopa were potentiated by carbidopa (50 mg/kg i.p.), and even 3-OMD levels in dialysate samples became detectable. Both OR-611 and Ro 40-7592 significantly further increased the DA and DOPAC efflux from striatum produced by L-dopa. This increase was more pronounced when carbidopa was added to the treatment. OR-611 did not modify the effect of L-dopa or carbidopa/L-dopa on dialysate HVA levels, whereas Ro 40-7592 markedly reduced those levels. Both OR-611 and Ro 40-7592 very clearly suppressed dialysate 3-OMD levels produced by carbidopa/L-dopa. Ro 40-7592 was more effective than OR-611 in potentiating the effects of L-dopa or carbidopa/L-dopa. These in vivo data show that the new COMT inhibitors markedly inhibit the O -methylation of L-dopa and increase its availability to brain, which is reflected as increased DA formation. A significant effect can be achieved even by inhibiting only the peripheral COMT activity. The data suggest that COMT inhibitors may be of clinical importance as adjuncts in the treatment of Parkinson's disease.     

Dose-dependent effects of FK506 on neuroregeneration in a rat model

This study explored the effects of different doses of FK506 on peripheral nerve regeneration, to determine whether neuroregeneration could be enhanced without the toxicity of systemic immunosuppression. In the first part of the study, subimmunosuppressive doses of FK506 were determined by examining skin allograft survival in a rat model. Full-thickness skin grafts (2 cm2) from Wistar rats were grafted to recipient Lewis rats. The procedure was performed for six groups (n = 6). The control group received no FK506, and the other five groups received daily doses of FK506 of 0.125, 0.25, 0.5, 1.0, or 2.0 mg/kg. Animals that received 2.0 mg/kg FK506 per day exhibited complete skin graft take, whereas all other groups demonstrated complete rejection. After determination of the immunosuppressive dose of FK506, the neuroregenerative effects of different doses of FK506 were explored by assessing nerve regeneration in 80 rats after tibial nerve transection and repair. The control group received no FK506, whereas the other four groups were given daily doses of FK506 of 0.25, 0.5, 1.0, or 2.0 mg/kg. Rats were euthanized at three time points (25, 30, and 35 days), to fully investigate the effects of different FK506 dosing regimens on neuroregeneration. Histomorphometric analyses performed on postoperative days 30 and 35 demonstrated statistically significant improvements in neuroregeneration with subimmunosuppressive FK506 doses of 0.5 and 1.0 mg/kg per day. Therefore, the study demonstrated that neuroregeneration was enhanced at low doses of FK506 that were not sufficient to prevent skin allograft rejection.     

免疫抑制剂FK506对大鼠移植性肝癌肿瘤生长和脾转移的影响

目的观察在大鼠移植性肝癌模型中不同FK506剂量下肿瘤的大小和侵润以及脾脏转移情况,用以评价FK506对肝癌细胞增殖和转移的情况。方法在Wistar大鼠肝左叶种植walker-256细胞株成瘤的癌组织块以构建转移性肝癌模型,54只大鼠随机分为FK506低剂量组（0.5 mg/kg.d）,正常剂量组（1.0 mg/kg.d）和空白对照组,测量肿瘤大小,通过病理组织学观察7、14、21 d脾脏转移发生率和中位数。结果与空白组相比,第14、21天两个剂量组肝癌体积明显增大（P〈0.05）;同时也出现了脾转移,第14天FK506各组转移中位数均不同。根据病理组织学,各组脾转移的发生率没有明显性差异。结论用药早期,脾脏还存在免疫功能,免疫抑制剂不能完全压制抗肿瘤效应。低剂量FK506也许有利于减少癌症脾转移,仍需进一步的探讨FK506低剂量长期使用在肝癌复发和转移的作用。     

Effects of dextromethorphan on dopamine dependent behaviours in rats

Dextromethorphan, a noncompetitive blocker of N-methyl-D- aspartate (NMDA) type of glutamate receptor, at 7.5-75 mg/kg, ip did not induce oral stereotypies or catalepsy and did not antagonize apomorphine stereotypy in rats. These results indicate that dextromethorphan at 7.5-75 mg/kg does not stimulate or block postsynaptic striatal D2 and D1 dopamine (DA) receptors. Pretreatment with 15 and 30 mg/kg dextromethorphan potentiated dexamphetamine stereotypy and antagonised haloperidol catalepsy. Pretreatment with 45, 60 and 75 mg/kg dextromethorphan, which release 5-hydroxytryptamine (5-HT), however, antagonised dexamphetamine stereotypy and potentiated haloperidol catalepsy. Apomorphine stereotypy was not potentiated or antagonised by pretreatment with 7.5-75 mg/kg dextromethorphan. This respectively indicates that at 7.5-75 mg/kg dextromethorphan does not exert facilitatory or inhibitory effect at or beyond the postsynaptic striatal D2 and D1 DA receptors. The results are explained on the basis of dextromethorphan (15-75 mg/kg)-induced blockade of NMDA receptors in striatum and substantia nigra pars compacta. Dextromethorphan at 15 and 30 mg/kg, by blocking NMDA receptors, activates nigrostriatal dopaminergic neurons and thereby potentiates dexampetamine stereotypy and antagonizes haloperidol catalepsy. Dextromethorphan at 45, 60 and 75 mg/kg, by blocking NMDA receptors, releases 5-HT and through the released 5-HT exerts an inhibitory influence on the nigrostriatal dopaminergic neurons with resultant antagonism of dexampetamine stereotypy and potentiation of haloperidol catalepsy.     

Effects of narcotic antagonists on L-dopa reversal of reserpine-induced catalepsy and blepharoptosis in mice

Reserpine (1 mg/kg, i.p.) induced catalepsy and blepharoptosis in mice which were readily reversed by the administration of L-dopa (300 mg/kg, i.p.). The administration of the pure narcotic antagonists naloxone (10 mg/kg, i.p.) and naltrexone (1 mg/kg, i.p.) significantly potentiated L-dopa reversal of reserpine-induced catalepsy. Lower doses of the narcotic antagonists did not significantly alter this reversal. The L-dopa reversal of blepharoptosis was not significantly altered by either naloxone or naltrexone. These results indicate that while opiate receptors may be involved in L-dopa reversal of catalepsy, they may not have a role in the alteration of blepharoptosis.     

Effect of BR-16A (Mentat), a polyherbal formulation on drug-induced catalepsy in mice

Parkinson's disease (PD) is a neurodegenerative disease characterized by the selective loss of dopamine (DA) neurons of the substantia nigra pars compacta (SNc). The events, which trigger and/or mediate the loss of nigral DA neurons, however, remain unclear. Neuroleptic-induced catalepsy has long been used as an animal model for screening drugs for Parkinsonism. Administration of haloperidol (1 mg/kg, ip) or reserpine (2 mg/kg, ip) significantly induced catalepsy in mice. BR-16A (50 and 100 mg/kg, po), a polyherbal formulation or ashwagandha (50 and 100 mg/kg, po), significantly reversed the haloperidol or reserpine-induced catalepsy. The results indicate that BR-16A or ashwagandha has protective effect against haloperidol or reserpine-induced catalepsy and provide hope that BR-16A could be used in preventing the drug-induced extrapyramidal side effects and may offer a new therapeutic approach to the treatment of Parkinson's disease.     

Chorionic gonadotropin-like proteins in the obplacental giant cells of the rabbit

Diabetes mellitus was induced in 40 male C57BL6 mice by injection of a low dose of streptozocin (45 mg/kg body weight) on 5 consecutive days. Twenty four of the mice were immunosuppressed by administration of 1.5 mg FK506/kg body weight daily for 10, 15, 18 and 24 days. Administration of FK506 almost completely inhibited the streptozocin-induced islet damage, and consequently glycaemia remained normal. In FK506-treated animals any inflammatory infiltrate was very sparse and was limited to the vascular pole of the islets. Immunocytochemical results demonstrated that infiltrating cells were Ia-immunoreactive, but were not activated. Ultrastructural observations confirmed the absence of B cell necrosis and degranulation in FK506-treated mice; the few infiltrating elements encountered did not contain phagocytic vesicles or show other signs of activation.     

Tacrolimus (FK506) Prevents Early Stages of Ethanol Induced Hepatic Fibrosis by Targeting LARP6 Dependent Mechanism of Collagen Synthesis

Tacrolimus (FK506) is a widely used immunosuppressive drug. Its effects on hepatic fibrosis have been controversial and attributed to immunosuppression. We show that in vitro FK506, inhibited synthesis of type I collagen polypeptides, without affecting expression of collagen mRNAs. In vivo, administration of FK506 at a dose of 4 mg/kg completely prevented development of alcohol/carbon tetrachloride induced liver fibrosis in rats. Activation of hepatic stellate cells (HSCs) was absent in the FK506 treated livers and expression of collagen α2(I) mRNA was at normal levels. Collagen α1(I) mRNA was increased in the FK506 treated livers, but this mRNA was not translated into α1(I) polypeptide. No significant inflammation was associated with the fibrosis model used. FK506 binding protein 3 (FKBP3) is one of cellular proteins which binds FK506 with high affinity. We discovered that FKBP3 interacts with LARP6 and LARP6 is the major regulator of translation and stability of collagen mRNAs. In the presence of FK506 the interaction between FKBP3 and LARP6 is weakened and so is the pull down of collagen mRNAs with FKBP3. We postulate that FK506 inactivates FKBP3 and that lack of interaction of LARP6 and FKBP3 results in aberrant translation of collagen mRNAs and prevention of fibrosis. This is the first report of such activity of FK506 and may renew the interest in using this drug to alleviate hepatic fibrosis.     

Intravenously injected FK506 failed to inhibit hippocampal calcineurin

FK506 (tacrolimus) is known as an inhibitor for calcineurin and is used in numerous research fields. It is not clear whether intravenously injected FK506 inhibits neuronal calcineurin. We measured the calcineurin activities of normal and postischemic rat hippocampi after intravenous injection of FK506 (3 mg/kg). Intravenously injected FK506 had no inhibitory effect on calcineurin activity in the hippocampi of normal and postischemic rats, whereas FK506 inhibited the calcineurin in vitro (purified enzyme, hippocampal homogenate, and hippocampal slice culture homogenate). Thus, it is considered that intravenously injected FK506 does not act on intraneuronal calcineurin and that several effects of FK506 are not due to the inhibition of neuronal calcineurin.     

Effect of FK506 on osteoinduction by recombinant human bone morphogenetic protein-2

FK506 is an immunosuppressant that is used widely in organ transplantation, and it has recently been recognized as effective for promoting the growth of bone grafts [J. Bone Miner. Res. 15 (2000) 1147]. In this study, we evaluated the influence of FK506 on osteoinduction by recombinant human bone morphogenetic protein-2 (rhBMP-2) using atelopeptide type I collagen as a carrier. We administered FK506 (1 mg/kg/day intramuscularly) on days -2 to 0, -2 to 7, and -2 to sacrifice. rhBMP-2 was implanted into the calf muscle of Wistar rats (thirty per group) and the implant was sampled on days 7, 14, and 21. Radiographic evaluation, histological examination, and biochemical analysis were performed. It was found that FK506 promoted the early stage of osteoinduction after short-term administration. However, long-term administration of this agent accelerated both bone formation and bone resorption. In order to use FK506 effectively for promoting bone growth, we must further examine the appropriate dose, method, and period of administration.     

Local FK506 dose-dependent study using a novel three-dimensional organotypic assay

Local administration of FK506, an FDA approved immunosuppressant with neuroregenerative properties, is a promising technique to achieve improved peripheral nerve regeneration while preventing the side effects associated with the systemic administration of this drug. Although considerable research has been devoted to the development of clinically suitable systems for local delivery of FK506 to the site of nerve injury and repair, the optimal dose of FK506 for enhancement of axon regeneration in the peripheral nerve has not yet been established. To this end, we devised a three-dimensional (3D) organotypic assay capable of mimicking the peripheral nerve. This assay consisted of a neonatal rat dorsal root ganglion (DRG) extending its neurites into the native peripheral nerve scaffold provided by an acellular nerve allograft (ANA). A novel 3D compartmented cell culture system was adapted from the 3D organotypic assay to achieve local delivery of FK506 just to the growing neurites in vitro and establish the required local dose of FK506 for peripheral nerve regeneration. A bimodal dose response was observed by culturing the entire DRG–ANA construct with media containing different concentrations of FK506. Low drug concentration of 1 pg/ml and high drug concentration of 100 ng/ml lead to the longest neurite extension in vitro. Furthermore, regardless of the FK506 concentration, concentrating the drug to the growing neurites resulted in significant increase in both neurite extension and neurite density, an effect that was not observed with the FK506 delivery to both neurites and neural cell bodies within DRG. The findings in this study provide valuable insight into the optimal local dose of FK506 for peripheral nerve regeneration. Furthermore, for the first time, this study suggests the potential interaction of FK506 with axons at the level of the growth cone.     

Inhibition of anticonvulsant action of carbamazepine by aminophylline and caffeine in rats

Interaction of two well known methyl xanthines, aminophylline--an antiasthmatic agent--and caffeine--commonly present in beverages, on the seizure protective ability of carbamazepine (CBZ) against electrically and chemically induced seizures in rats was investigated. Aminophylline (75 mg/kg, ip) did not alter the activity of CBZ (10 mg/kg, ip; ED100) on maximal electroshock seizures while dose dependent antagonism of CBZ efficacy was seen at 100 and 150 mg/kg, ip. Similar effects were observed with caffeine (200 and 250 mg/kg, ip). At the highest tolerated doses, aminophylline (150 mg/kg, ip) and caffeine (250 mg/kg, ip) produced antagonism of CBZ protection against pentylenetetrazole seizures. These observations support the possibility that the antagonism due to the interaction of these drugs could be related to their action at adenosine receptor sites in the brain.     

Peripheral urocortin inhibits gastric emptying and food intake in mice: differential role of CRF receptor 2

Intraperitoneal urocortin inhibits gastric emptying and food intake in mice. We investigated corticotropin-releasing factor receptor (CRF-R) subtypes involved in intraperitoneal urocortin actions using selective CRF-R antagonists. Gastric emptying was measured 2 h after a chow meal, and food intake was measured hourly after an 18-h fast in mice. Urocortin (3 microg/kg ip) inhibited gastric emptying by 88%. The CRF-R1/CRF-R2 antagonist astressin B (30 microg/kg ip) and the selective CRF-R2 antagonist antisauvagine-30 (100 microg/kg ip) completely antagonized urocortin action, whereas the selective CRF-R1 antagonist CP-154,526 (10 mg/kg ip) had no effect. Urocortin (1-10 microg/kg ip) dose dependently decreased the 2-h cumulative food intake by 30-62%. Urocortin (3 microg/kg)-induced hypophagia was completely antagonized by astressin B (30 microg/kg ip) and partially (35 and 31%) by antisauvagine-30 (100 or 200 microg/kg ip). The CRF-R1 antagonists CP-154,526 or DMP904 (10 mg/kg ip) had no effect. Capsaicin did not alter urocortin-inhibitory actions while blocking the satiety effect of intraperitoneal CCK. These data indicate that intraperitoneal urocortin-induced decrease in feeding is only partly mediated by CRF-R2, whereas urocortin action to delay gastric emptying of a meal involves primarily CRF-R2.     

Sustained QT prolongation induced by tacrolimus in guinea pigs.

Recently, clinical cases have been reported of QT prolongation and torsades de pointes associated with the use of tacrolimus (FK506). We examined the relationship between QTc prolongation and the pharmacokinetics of FK506 in guinea pigs in order to evaluate the arrhythmogenicity of FK506 in comparison with quinidine (QND). FK506 (0.1 or 0.01 mg/hr/kg) or QND (30 mg/hr/kg) was intravenously infused to guinea pigs and time profiles of drug concentration in blood and QTc interval were examined during and after infusion. Both FK506 and QND evoked a significant QTc prolongation, and the dose-response relationship showed an anti-clockwise hysteresis, FK506-induced QTc prolongation persisted throughout the duration of the experiment despite a decline in the plasma FK506 concentration, whilst QND-induced QTc prolongation disappeared as plasma concentrations decreased. FK506 induced a sustained QTc prolongation in guinea pigs at drug concentrations in blood that correspond to its therapeutic range in human, suggesting that it might be of clinical significance to monitor the electrocardiogram, especially when patients have congenital or acquired QT-prolonging risk factors.     

Sodium selenite stimulates neurobehavior and neurochemical activities in rats

The effect of 0.05, 0.1, and 0.2 mg sodium selenite/kg body weight ip on the activities of neurobehavioral, acetyl cholinesterase, monoamine oxidase, and the content of dopamine and its metabolites in circadian rhythm centers of male Wistar rats was studied after 7 d of treatment. The results show an appreciable increase in locomotion, stereo-events, distance traveled, and average speed at the dose of 0.1 and 0.2 mg sodium selenite/kg. The data have shown hyperactivity of animals with various doses of sodium selenite, and it was significant and dose-dependent after 3 d of treatment. The activity of acetylcholinesterase (AChE) was inhibited dose dependently, and it was significant in preoptic area with 0.1 or 0.2 mg sodium selenite/kg. Conversely, in the posterior hypothalamus its activity was significantly elevated with the dose of 0.2 mg sodium selenite/kg, but its alteration in brain stem was not significant. Monoamine oxidase (MAO) activity was increased in preoptic area with the dose of 0.1 mg sodium selenite/kg, but its alteration in posterior hypothalamus and brain stem was not significant. The content of dopamine (DA), 3,4-dihydroxyphenyl acetic acid (DOPAC), and homovanilic acid (HVA) was elevated dose dependently and it was significant with the doss of 0.1 and 0.2 mg sodium selenite/kg, but the content of DOPAC and HVA in posterior hypothalamus was not significant with the dose of 0.1 mg sodium selenite/kg.     

Hepatoprotective effect of tocopherol against isoniazid and rifampicin induced hepatotoxicity in albino rabbits

Antitubercular drug induced hepatotoxicity is a major hurdle for an effective treatment of tuberculosis. The present study was undertaken to assess the hepatoprotective potential of tocopherol (50 mg/kg and 100 mg/kg, ip) and to compare it with cimetidine (120 mg/kg, ip). Hepatotoxicity was produced by giving isoniazid (INH, 50 mg/kg, po) and rifampicin (RMP, 100 mg/kg, po) combination to albino rabbits for 7 days. Assessment of liver injury was done by estimating levels of alanine transaminase (ALT) and argininosuccinic acid lyase (ASAL) in serum and by histopathological examination of liver. Results revealed that pretreatment with high dose of tocopherol (100 mg/kg) prevented both biochemical as well as histopathological evidence of hepatic damage induced by INH and RMP combination. Moreover, tocopherol (100 mg/kg) was found to be a more effective hepatoprotective agent as compared to cimetidine.     

FK 506 and aminoguanidine suppress iNOS induction in orthotopic corneal allografts and prolong graft survival in mice.

The aim of this study was to compare the effectiveness of immunosuppressant FK 506 and the specific inhibitor of inducible nitric oxide synthase (iNOS) aminoguanidine (AG) in prevention of corneal graft rejection and to investigate the iNOS expression in the rejection process. Orthotopic corneal allografting in mice was performed (C57BL/10; H-2(b) to BALB/c; H-2(d)). FK 506 (0.3 mg/kg per day) or AG (100 mg/kg per day) was injected intraperitoneally for 4 weeks. Grafted mice without therapy served as controls. Immunohistological evaluation of iNOS-positive cells and macrophage infiltration in grafts 27th day after grafting was performed. Within 4 weeks FK 506 prevented graft rejection in 71% and AG in 57% of animals compared to 29% of clear grafts in controls. A significant proportion of iNOS-positive cells was detected in the rejected grafts of the control and AG-treated groups. The treatment with FK 506 resulted in the inhibition of iNOS expression to a high degree in the rejected corneas. Non-rejected corneas of all groups and non-transplanted corneas exhibited no iNOS-positive cells. A massive infiltration of macrophages was detected in the rejected grafts, whereas non-rejected grafts exhibited only slight infiltration of macrophages. The presented data suggest that overexpression of iNOS and/or activation of iNOS is one of the several influential factors that contribute to the rejection process and that iNOS suppression delays corneal allograft rejection. FK 506 and AG are effective drugs in preventing corneal allograft rejection. Higher beneficial effect of FK 506 on graft survival could be explained by its well-known selective T-cell immunosuppression.     

Pharmacological responses to acute morphine administration in normotensive Wistar-Kyoto and spontaneously hypertensive rats

The effect of acute administration of morphine on analgesia, hyperthermia, hypothermia and catalepsy was determined in spontaneously hypertensive (SH) rats and normotensive Wistar-Kyoto (WKY) rats. A greater analgesic and hyperthermic response to morphine was observed in SH rats than in WKY rats. A dose of morphine (50 mg/kg ip) which produced hypothermia in WKY rats produced pronounced hyperthermia in SH rats. The cataleptic response to morphine was lower in SH rats. The cataleptic response to morphine was lower in SH rats than in WKY rats. The brain and plasma levels of morphine in SH rats were significantly lower as compared to the WKY rats at any dose of morphine used but the ratio of brain to plasma did not differ. It is concluded that SH rats exhibit altered sensitivity to morphine in comparison with their normotensive counterparts.     

FK506 accelerates functional recovery following nerve grafting in a rat model.

The sometimes dramatic and permanent functional deficits that result from severe peripheral nerve injuries provide compelling incentives to identify exogenous agents that may expedite axonal regrowth and avoid prolonged denervation of end organs. The purpose of this study was to identify, whether the regular systemic administration of tacrolimus (FK506) or cyclosporin A (CsA) would influence the speed and efficiency of nerve regeneration through short nerve grafts. A total of 35 Buffalo rats each received a 2-cm posterior tibial nerve graft and were randomized to one of three experimental groups. Group I animals were left untreated, group II received daily CsA (5 mg/kg intraperitoneally), and group III received daily FK506 (1 mg/kg intraperitoneally). Walking tracks were obtained starting 3 weeks after graft placement and continuing biweekly for the next 7 weeks. FK506-treated animals fully recovered hindlimb function 7 days earlier than CsA-treated animals or untreated control animals. Regenerated nerves from one-half of each treatment group were harvested for histomorphometric analysis at 7 weeks, shortly after recovery was complete in the FK506-treatment group but not in the other two groups, and once again at 10.5 weeks when recovery of function had stabilized in all groups. At 7 weeks, FK506-treated animals had significantly greater fiber density and percentage of neural tissue per nerve and a significantly larger population of mature, myelinated fibers in comparison with either CsA-treated or untreated animals. The authors concluded that the daily, systemic administration of low-dose FK506 facilitates peripheral nerve recovery and regeneration after nerve grafting.