Pharmacological profile of FK881(ASP6537), a novel potent and selective cyclooxygenase-1 inhibitor.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are now understood to fall into one of two agent classes in clinical use. Traditional NSAIDs inhibit both cyclooxygenases-1 and 2 (COX-1, 2), which act as key enzymes catalyzing the same reaction in the production of prostaglandins (PGs), while the second class of NSAIDs selectively inhibit COX-2. Inhibition of the inducible COX-2 isoform is believed to be responsible for the therapeutic effects of NSAIDs, such as anti-inflammatory, analgesic, and antipyretic effects, while COX-1 inhibition results in side-effects on the gastrointestinal (GI) system. In the present study, however, we changed this notion that inhibiting only COX-1 causes adverse effects. We discovered FK881, a specific COX-1 inhibitor which exhibits a 650-fold ratio for human whole blood COX-1/COX-2 and rats in vivo. In rats, FK881 dose dependently inhibited carrageenan-induced paw edema (ED30: 22 mg/kg; diclofenac ED30: 3.6 mg/kg, rofecoxib ED30: 26 mg/kg) and paw swelling associated with adjuvant arthritis (ED50: 17 mg/kg; diclofenac ED50: 1.4 mg/kg, rofecoxib ED50: 1.8 mg/kg). Further, FK881 dose dependently inhibited acetic acid-induced writhing in mice (ED50: 19 mg/kg; diclofenac ED50: 14 mg/kg, rofecoxib ED50: >100mg/kg) and adjuvant arthritis hyperalgesia in rats (ED50: 1.8 mg/kg; diclofenac ED50: 1.0mg/kg, rofecoxib ED50: 0.8mg/kg). However, unlike traditional NSAIDs, GI tolerability was improved, although the antipyretic effect of FK881 was weak (NOEL: >320 mg/kg; diclofenac NOEL: <1mg/kg, rofecoxib NOEL: 100 mg/kg). These results suggest that FK881 may be useful in treating symptoms of rheumatoid arthritis and osteoarthritis.

Prevention of renal interstitial fibrosis via histone deacetylase inhibition in rats with unilateral ureteral obstruction.

Acute rejection following renal transplantation has become manageable with the introduction of calcineurin inhibitors, FK506 and cyclosporine A. However, chronic allograft dysfunction accompanied by renal interstitial fibrosis, which induces graft loss, remains unresolved. Here, we evaluated the effect of FR276457, a pan-histone deacetylase (HDAC) inhibitor, on interstitial fibrosis in the injured kidneys of a rat model of unilateral ureteral obstruction. The injured kidneys, harvested on Day 14 following the operation, showed progression of interstitial fibrosis, increases of hydroxyproline contents, and mRNA expression of collagen type Ialpha1 and monocyte chemotactic protein 1 (MCP-1). However, these changes were found to be prevented with daily oral administration of FR276457. In addition, given that MCP-1 is believed to contribute to progressive fibrosis, we investigated the direct effect of FR276457 on MCP-1 production by activated THP-1 cells in vitro. Results showed that FR276457 administration decreased MCP-1 production in these cells in a concentration-dependent manner. Findings from the present study suggested that a pan-HDAC inhibitor may exert a prophylactic effect against renal interstitial fibrosis by inhibiting MCP-1 production.

ASP4000, a slow-binding dipeptidyl peptidase 4 inhibitor, has antihyperglycemic activity of long duration in Zucker fatty rats.

ASP4000 ((2S)-1-{[(1R,3S,4S,6R)-6-hydroxy-2-azabicyclo[2.2.1]hept-3-yl]carbonyl}-2-pyrrolidinecarbonitrile hydrochloride) is a novel, potent and selective dipeptidyl peptidase 4 (DPP IV, EC 3.4.14.5) inhibitor (Keiko Tanaka-Amino et al. in Eur J pharmacol 59:444-449, 2008). The aim of the present study was to characterize the kinetic profile of and identify the long duration effect of the antihyperglycemic activity of ASP4000. ASP4000 was found to inhibit human recombinant DPP4 activity with a K(i) of 1.05 nM, a k(on) value of 22.3 x 10(5) M(-1) s(-1), and a k (off) of 2.35 x 10(-3) M(-1) s(-1), with higher affinity than that of vildagliptin. The kinetic studies indicate that both the formation and dissociation of ASP4000/DPP4 complex were faster than those of vildagliptin, and that ASP4000 slow-bindingly inhibits DPP4 with a different mode of inhibition than vildagliptin. In addition, ASP4000 augmented the insulin response and ameliorated the glucose excursion during the oral glucose tolerance test in Zucker fatty rats at 4 h post dosing. ASP4000 is expected to be a promising, long duration DPP4 inhibitor for type 2 diabetes.

Constitutive increase in active GLP-1 levels by the DPP4 inhibitor ASP4000 on a new meal tolerance test in Zucker fatty rats.

Glucagon-like peptide-1 (GLP-1), an incretin hormone, is essential for the regulation of insulin secretion and glucose homeostasis. GLP-1 is rapidly degraded by dipeptidyl peptidase 4 (DPP4); therefore, DPP4 inhibitors are considered to be a novel class of oral antihyperglycemic agents. These agents are currently under development as treatments for type 2 diabetes. Normally, oral glucose tolerance tests are used for evalating glucose-lowering efficacy, but the augmentation of active GLP-1 via DPP4 inhibition in this test was transient. It has been proposed that the secretion of GLP-1 is regulated by the rate of entry of nutrients into the small intestine; therefore, we have established the new meal tolerance test method using solid diet. This model allows for the continuous monitoring of active GLP-1 secretion after food intake. ASP4000 is an orally effective inhibitor of DPP4 that greatly augments meal-stimulated circulating levels of active GLP-1 constitutively and improves hyperglycemia. Acarbose improved glucose tolerance in the test to a degree similar to that of the DPP4 inhibitor. Our new meal tolerance test is useful for evaluating postprandial hyperglycemia and could be an excellent model for studying the secretion of active GLP-1 via the inhibition of DPP4.

Effect of the immunosuppressant histone deacetylase inhibitor FR276457 in a canine renal transplant model.

The histone deacetylase (HDAC) inhibitor FR276457, a hydroxamic derivative, was identified during chemical library screening and was found to exhibit potent inhibitory effects on the activity of mammalian HDACs. It has been shown that FR276457 exhibited marked immunosuppressive effects in a rat heterotopic cardiac transplant model. To predict clinical efficacy of FR276457, we investigated the inhibitory effect of the proliferation of Jurkat cells in vitro and immunosuppressive effect of orally administered FR276457 on allograft rejection as a monotherapy or in combination with tacrolimus (0.04 mg/kg) injected intramuscularly (i.m.) in a canine renal transplant model. Animal survival, the plasma creatinine level, and histopathology were evaluated. FR276457 inhibited the proliferation of Jurkat cells probably by targeting activity of NF-kappaB. FR276457 prolonged the median survival time (MST) of transplanted grafts from 11.5 days (untreated group) to 29.0 days (FR276457-treated group). FR276457 administered 1 mg/kg twice a day in combination with tacrolimus prevented allograft rejection. In addition, a dose of 1.5 mg/kg twice a day or 5.0 mg/kg once a day prolonged the MST from 18 days (control group) to >73 or >90 days, respectively. Histopathological analysis showed that FR276457 suppressed the score for mononuclear cell infiltration and vasculitis. In conclusion, the HDAC inhibitor FR276457 inhibited the proliferation of T cell line established from human in vitro. And more, FR276457 clinically prolonged allograft survival when administered as a monotherapy, and had additive or synergistic effects when combined with tacrolimus with the canine renal transplant model. These results showed HDAC inhibitor is a promising biological target for treatment in transplant field.

Thirteen-month inhibition of aldose reductase by zenarestat prevents morphological abnormalities in the dorsal root ganglia of streptozotocin-induced diabetic rats.

The dorsal root ganglia (DRG) have been identified as the target tissue in diabetic somatosensory neuropathy. It has been reported that, in the chronically diabetic state, DRG sensory neurons may undergo morphological changes. In this study, we examined the effect of zenarestat, an aldose reductase inhibitor, on the morphological derangement of the DRG and the sural nerve of streptozotocin-induced diabetic rats (STZ rats) over a 13-month period. The cell area of the DRG in STZ rats was smaller than that in normal rats. A decrease in fiber size was apparent in the sural nerve of the STZ rats, and the fiber density was greater. These morphological changes were reversed in zenarestat-treated STZ rats. The data suggest that, in peripheral sensory diabetic neuropathy, hyperactivation of the polyol pathway induces abnormalities not only in peripheral nerve fiber, but also in the DRG, which is an aggregate of primary sensory afferent cell bodies.

Tacrolimus (FK506) suppresses rt-PA-induced hemorrhagic transformation in a rat thrombotic ischemia stroke model.

The aim of this study was to evaluate the effect of tacrolimus on recombinant tissue-plasminogen activator (rt-PA)-induced hemorrhagic transformation, and to characterize its suppressive action for hemorrhage. Thrombotic occlusion of the middle cerebral artery (MCA) was induced by photochemical reaction in spontaneously hypertensive rats, and hemorrhagic scores and brain damage were measured 24 h after MCA occlusion. Administration of rt-PA 3 h after MCA occlusion significantly worsened spontaneous hemorrhagic changes and tended to aggravate brain damage. Hematoma was observed in 7 of 15 rats treated with rt-PA, and 0 of 15 rats in the control group. Tacrolimus alone administered intravenously 3 h after MCA occlusion did not produce any hemorrhagic changes. The combined treatment of tacrolimus followed by rt-PA significantly decreased the incidence of hematoma and brain damage in comparison with that of the rt-PA treated group. Permeability of the blood-brain-barrier (BBB) detected by extravasations of Evans blue was investigated 6 h after MCA occlusion, as was the integrity of microvascular endothelial cells as determined by immunohistochemical assessment of the prevalence of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31). Combined treatment of rt-PA with tacrolimus reduced the rt-PA-induced extravasation of Evans blue and preserved CD31-positive cells in the ischemic hemisphere. Thus, tacrolimus was able to reduce the rt-PA-induced hemorrhagic transformation, which might be due to the protective effects on cerebral microvascular endothelial cells after thrombotic cerebral ischemia during the acute phase of cerebral ischemia. In conclusion, the combination of rt-PA with tacrolimus may be useful for decreasing the risk of thrombolytic therapy.

Efficacy of oral treatment with tacrolimus in the renal transplant model in cynomolgus monkeys.

Many studies have examined the efficacy of tacrolimus in rats and dogs, but few have reported its evaluation in cynomolgus monkeys. The aim of this study was to clarify the efficacy of tacrolimus in a cynomolgus monkey renal transplant model based on the efficacy of various doses. Monkeys that had undergone renal transplant were treated with a vehicle or 0.5, 1.0, or 2.0 mg/kg of tacrolimus by oral administration. Tacrolimus administration prolonged animal survival in a dose-dependent manner, and the median survival time (MST) was 11, 21, and >90 days for the 0.5, 1.0, and 2.0 mg/kg tacrolimus groups, respectively. The MST of the vehicle group was 6 days. Histopathological analyses of all transplanted kidneys were also performed. Typical pathological findings of acute rejection were observed in both the vehicle and tacrolimus (0.5 and 1.0 mg/kg)-treated groups. Only limited mononuclear cell infiltration and hemorrhage were present in the tacrolimus (2.0 mg/kg)-treated group. In conclusion, 2.0 mg/kg was considered to be a therapeutic dose in this model, and 0.5 or 1.0 mg/kg could be used for a study when efficacy of a new compound is evaluated in a combination therapy with tacrolimus.

Effect of a new immunosuppressant histon deacetylase (HDAC) inhibitor FR276457 in a rat cardiac transplant model.

Histone deacetylase (HDAC) is a known modulator of gene transcription, and the immunosuppressive activity of HDAC inhibitors has been demonstrated in recent several reports. In this study, the HDAC inhibitor FR276457, a hydroxamic derivative, was found to have a similar inhibitory effect on all mammalian HDACs tested, but no isozyme selectivity. Both FR276457 and tacrolimus exerted an immunosuppressive effect on in vitro rat splenocyte proliferation stimulated with Concanavalin A. Next, the effect of FR276457 on allograft rejection when administered either as a monotherapy or in combination with tacrolimus was investigated in a rat heterotopic cardiac transplant model. Orally administered FR276457 prolonged the median survival times (MST) of the transplanted grafts in the vehicle group from 6 d to 17 or 21 d at doses of 20 or 40 mg/kg, respectively. Histopathological analysis showed the structures of the myocardium were not affected, but interstitial cellular infiltration could not be suppressed completely. Tacrolimus (0.032 mg/kg) prolonged allograft MST to 16 d. FR276457, when combined with tacrolimus, prevented allograft rejection at a dose lower than that of the monotherapy. The combination dose prolonged the MST in the groups treated with 10 and 20 mg/kg to >28 d, and cellular infiltration was suppressed completely. In conclusion, this study demonstrated that the oral administration of HDAC inhibitor FR276457 can prevent allograft rejection as a monotherapy, and has additive or synergistic effects when combined with tacrolimus.

FK1706, a novel non-immunosuppressive immunophilin ligand, modifies the course of painful diabetic neuropathy.

FK1706, a derivative of FK506, is a non-immunosuppressive immunophilin ligand with significant neurotrophic activity mediated via FKBP-52 and the RAS/RAF/MAPK signaling pathway. Here, we tested the effect of FK1706 on painful diabetic neuropathy in rat model of diabetes induced by streptozotocin (STZ). FK1706 ameliorated mechanical allodynia in this model at doses over 0.32 mg/kg, p.o., even if treatment was initiated after neuropathy was established, and did not affect plasma glucose levels. Furthermore, this improvement continued at least 4 weeks after the last administration. In morphological analysis, FK1706 treatment also restored intraepidermal nerve fiber density in footpad skin to almost normal levels. Gabapentin also improved mechanical allodynia in the same model, but efficacy disappeared the day after administration stopped. Allodynia responses were potentiated by co-administration of both compounds. Thus, FK1706 ameliorated painful diabetic neuropathy via a different mechanism from gabapentin and improved morphological outcomes, indicating that FK1706 improves painful diabetic neuropathy by modifying the underlying disease pathology.

Use of a serum-based antifungal susceptibility assay to predict the in vivo efficacy of novel echinocandin compounds.

In vitro susceptibility assays of antifungal activity do not always accurately predict in vivo efficacy. As well as having a clear clinical importance, the ability to predict efficacy is also essential for effective screening of novel drug compounds. Initial screening of novel compounds must often be based on in vitro data. The present report describes the use of serum-MIC, an in vitro test of antifungal susceptibility, to accurately predict in vivo efficacy of echinocandin drugs in a mouse model of disseminated candidiasis. The basis of the serum-MIC method was to measure the inhibitory activity of a test compound against Candida albicans hyphal growth in the presence of pooled mouse serum. For 13 previously uncharacterized echinocandin compounds, as well as for the known echinocandin drugs, micafungin and caspofungin, serum-MIC determinations were shown to give better correlation to efficacy in the animal model than conventional, CLSI standard, in vitro antifungal susceptibility tests. The most accurate prediction of efficacy was obtained when the serum-MIC was adjusted in relation to the serum concentration at 30 min post-treatment. Furthermore, when the efficacy of micafungin was determined by measuring C. albicans kidney burden in the mouse model of infection, the adjusted serum-MIC consistently reflected the effective serum concentrations. Our data indicate that determination of serum-MIC values will facilitate prediction of the in vivo potency of new antifungal compounds such as novel echinocandins.

FK1706, a novel non-immunosuppressant neurophilin ligand, ameliorates motor dysfunction following spinal cord injury through its neuroregenerative action.

Injured spinal cord axons fail to regenerate in part due to a lack of trophic support. While various methods for replacing neurotrophins have been pursued, clinical uses of these methods face significant barriers. FK1706, a non-immunosuppressant neurophilin ligand, potentiates nerve growth factor signaling, suggesting therapeutic potential for functional deficits following spinal cord injury. Here, we demonstrate that FK1706 significantly improves behavioral outcomes in animal models of spinal cord hemisection and contusion injuries in rats. Furthermore, we show that FK1706 is effective even if administration is delayed until 1 week after injury, suggesting that FK1706 has a reasonable therapeutic time-window. Morphological analysis of injured axons in the dorsal corticospinal tract showed an increase in the radius and perimeter of stained axons, which were reduced by FK1706 treatment, suggesting that axonal swelling and retraction balls observed in injured spinal cord were improved by the neurotrophic effect of FK1706. Taken together, FK1706 improves both behavioral motor function and the underlying morphological changes, suggesting that FK1706 may have therapeutic potential in meeting the significant unmet needs in spinal cord injury.

Novel method for selecting immunosuppressive histone deacetylase (HDAC) inhibitors with minimal thrombocytopenia.

Histone deacetylase (HDAC) inhibitors repress interleukin-2 (IL-2) gene expression in T cells and possess immunosuppressive activity in vivo. In addition to its immunosuppressive activity, HDAC inhibitors block GATA binding protein-1 (GATA-1) gene expression in megakaryocytes and elicit thrombocytopenia. In this report we state that for a given immunosuppressive dose of HDAC inhibitor, the ratio of GATA-1 reporter gene activity relative to IL-2 reporter gene assay (G/I ratio of measured IC(50)) can be predictive of a HDAC inhibitor's thrombocytopenic effect. This study utilized nine HDAC inhibitors at a minimal effective dose in a rat heterotopic cardiac transplantation model and the resultant G/I ratios and platelet depletion rates were highly correlated (r=0.933). These results indicate that calculation of G/I ratio can be a novel method for selecting immunosuppressive HDAC inhibitor having minimal thrombocytopenic effect which will benefit the search for new immunosuppressants of greater safety and efficacy.

Direct comparison of the pharmacodynamics of four antifungal drugs in a mouse model of disseminated candidiasis using microbiological assays of serum drug concentrations.

The aim of this study was to compare the pharmacodynamics of the azole antifungal drugs fluconazole, itraconazole and ketoconazole, and the polyene antifungal amphotericin B, in a mouse model of disseminated Candida albicans infection. In order to directly compare effective serum concentrations of these antifungals, drug concentrations were assayed microbiologically by measuring inhibition of C. albicans mycelial growth (mMIC) in a mouse serum-based assay (serum antifungal titer). Efficacy in the mouse infection model was determined using an organ-based (kidney burden) endpoint. For all four drugs, the serum antifungal titers, 8 hr after administration of single doses of drugs at a range of drug concentrations, correlated closely with C. albicans kidney fungal burden in the mouse model. The results showed that determining serum antifungal titer may be used to accurately represent kidney fungal burden in a mouse model of disseminated candidiasis and allowed direct comparison of the pharmacodynamics of differing classes of antifungal drugs.

Mechanism of HDAC inhibitor FR235222-mediated IL-2 transcriptional repression in Jurkat cells.

Interleukin (IL)-2 is an essential cytokine in T cell proliferation and homeostasis. The importance of IL-2 down-regulation in preventing acute rejection in organ transplantation and the development of autoimmune diseases has been demonstrated by the therapeutic usefulness of the widely used immunosuppressants cyclosporine A and FK506. Recently, a histone deacetylase (HDAC) inhibitor, FR235222, has been shown to inhibit IL-2 gene expression and to possess immunosuppressive activity in vivo. To elucidate the inhibitory mechanism of FR235222 in IL-2 gene expression, we performed Affymetrix GeneChip analysis of activated Jurkat cells treated with or without FR235222. Here, we show that many NF-kappaB-regulated genes are transcriptionally down-regulated by FR235222 in activated Jurkat cells. Further, luciferase reporter assays revealed that FR235222 selectively inhibits NF-kappaB activity without impairing NF-AT or AP-1 at the concentrations at which it potently inhibits IL-2 promoter activation. These results indicate that FR235222 inhibits IL-2 gene expression via a different mechanism to CsA and FK506, and that FR235222 has the ability to inhibit NF-kappaB activity, which may be partly related to the potent inhibition of IL-2 gene expression by FR235222. Our findings may help our understanding of the molecular mechanism of the inhibition of IL-2 gene expression by HDAC inhibitors and provide insight into the development of more effective and safer new immunosuppressants.

Mechanisms of HDAC inhibitor-induced thrombocytopenia.

Histone deacetylase inhibitors (HDAC inhibitors) are an emerging class of anticancer agents. To elucidate the mechanism of HDAC inhibitor-induced thrombocytopenia, we focused on the effects of HDAC inhibitors on megakaryocyte differentiation and performed Affymetrix GeneChip analysis of human megakaryocytic HEL cells treated with or without HDAC inhibitors. Here, we report that GATA-1 and 10 haematopoietic factors (SCL, NF-E2, EKLF, Pleckstrin, Thrombin-R, LMO2, PU.1, Fli-1, AML1, and TCF11) are transcriptionally repressed by HDAC inhibitors in a similar pattern (R>0.98), and putative GATA-1-binding sites are found in almost all promoters of these genes. In addition, luciferase reporter assays reveal that mutations of GATA-1-binding sites in the GATA-1 promoter abolish its sensitivity to HDAC inhibitor-mediated down-regulation in HEL cells. Further, this report also asserts that HDAC inhibitor increases megakaryocyte counts and inhibits GATA-1 gene expression in rat spleen. Together, these results suggest that HDAC inhibitors inhibit GATA-1 gene expression by decreasing the transactivation function of GATA-1 itself, and that this may in turn lead to a delay in megakaryocyte maturation and finally cause thrombocytopenia. Our findings may help our understanding of the molecular mechanism of HDAC inhibitor-mediated GATA-1 transcriptional repression and to reduce the risk of HDAC inhibitor-induced thrombocytopenia.

Disruption of HDAC4/N-CoR complex by histone deacetylase inhibitors leads to inhibition of IL-2 gene expression.

Previous studies have shown that HDAC inhibitors selectively inhibit IL-2 gene expression, but the mechanism of this inhibition remains to be elucidated. It was recently reported that HDAC4, a component of the nuclear hormone receptor corepressor (N-CoR) complex, associates with the IL-2 promoter via the transcription factor myocyte enhancer factor 2 (MEF2). We therefore focused on the role of HDAC4/N-CoR complex in the transcriptional regulation of IL-2. Four approaches were used to characterize this role and to investigate the relation between the regulatory function of HDAC4/N-CoR complex and HDAC4-enzymatic activity: (i) HDAC4 silencing by RNA interference, (ii) overexpression of N-CoR repression domain 3 (RD3), (iii) overexpression of HDAC4 point mutants, and (iv) treatment with HDAC inhibitors. Here, we report that HDAC4 plays an essential role in IL-2 promoter activation, and that the formation of the HDAC4/N-CoR complex, which is closely related to HDAC4-enzymatic activity, might be involved in HDAC inhibitor-mediated inhibition of IL-2 gene expression. These observations indicate that the selective inhibition of HDAC4 or the interaction of HDAC4 with N-CoR is likely a potential target for the development of novel immunosuppressants.

Tacrolimus (FK506) limits accumulation of granulocytes and platelets and protects against brain damage after transient focal cerebral ischemia in rat.

We investigated the neuroprotective effect of tacrolimus (FK506) on the ischemia-reperfusion injury caused by transient focal brain ischemia induced by middle cerebral artery (MCA) occlusion for 60 min in rats. Neuronal damage visualized as a decrease of MAP2 immunoreactivity was observed in the cerebral cortex at 9 h after MCA occlusion and further expanded at 24 h. Hypoxic areas visualized with an immunohistochemical reaction for 2-nitroimidazole, a hypoxia marker (hypoxyprobe-1), and accumulation of granulocytes and platelets were also observed at 9 h and 24 h after MCA occlusion. Tacrolimus (1 mg/kg, i.v.), administered immediately after MCA occlusion, attenuated cortical damage and decreased the hypoxyprobe-1 positive area, as well as the number of granulocytes and platelets at 24 h after MCA occlusion. Immunohistochemical analysis showed that tacrolimus reduced the number of blood vessels positively stained for ICAM-1, E-selectin and P-selection. These results suggested that tacrolimus limited attachment of granulocytes and platelets to blood vessels by inhibiting the expression of adhesion molecules and protected neuronal tissue from hypoxic insults.

Therapeutic time window of tacrolimus (FK506) in a nonhuman primate stroke model: comparison with tissue plasminogen activator.

Tacrolimus (FK506), an immunosuppressive drug, has been shown to exert a potent neuroprotective activity when administered immediately after occlusion of the middle cerebral artery (MCA) in a nonhuman primate model of stroke. Here, we assessed the neuroprotective efficacy of tacrolimus with delayed treatment using the same model and compared with that of recombinant tissue plasminogen activator (rt-PA). Ischemic insult was induced by photochemically induced thrombotic occlusion of MCA in cynomolgus monkeys, and tacrolimus (0.2 mg/kg) and/or rt-PA (1.0 mg/kg) was intravenously administered 2 h after MCA occlusion. In another experiment, tacrolimus (0.1 mg/kg) was administered 4 h after MCA occlusion. Neurological deficits were monitored for 28 days after the ischemic insult and cerebral infarct volumes were measured with brain slices. With drug administration 2 h after the ischemic insult, tacrolimus significantly reduced neurological deficits and infarct volumes in the cerebral cortex without affecting the recanalization pattern in the MCA, however, rt-PA did not significantly improve neurological deficits or infarct volumes, even though it increased the recanalization rate of the occluded MCA. Combined treatment with tacrolimus and rt-PA exerted additional protection. Administration of tacrolimus 4 h after the ischemic insult still showed significant amelioration of neurological deficits. These results suggested that tacrolimus had a wider therapeutic time window than rt-PA in the nonhuman primate stroke model.

Temporal and topographic profiles of tissue hypoxia following transient focal cerebral ischemia in rats.

Intravascular accumulation of blood cells after brain ischemia-reperfusion can cause obstruction of cerebral blood flow and tissue hypoxia/ischemia as a consequence. In the present study, we examined temporal and topographic changes of tissue hypoxia/ischemia after occlusion of the middle cerebral artery (MCA) for 60 min in rats with immunohistochemical staining for hypoxia (2-nitroimidazole hypoxia marker: hypoxyprobe-1 adducts). Our results showed that tissue hypoxia expressed as positive staining for hypoxyprobe-1 adducts preceded neuronal degeneration. Platelets and granulocytes were detected close to the hypoxyprobe-1 adducts positive area. These results suggested that the hypoxic environment could persist even after reperfusion of MCA, because of vascular obstruction with accumulation of platelets and granulocytes.